TWI281473B - Biomarkers for liver diseases and method for using same - Google Patents

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1281473 玖、發明說明： 【發明所屬之技術領域】 本發明係關於肝臟疾病之生物標記分子及其檢測方法，係利 用自抗原師运方法師進出可用於檢測肝臟疾病之生物標記分 子，並利用這些生物標記分子發展為檢測套組，藉篩選檢體中自 體抗體或自體抗原的存在判斷是否罹患肝臟疾病。 【先前技術】 免疫系統若功能不健全，便會產生免疫性疾病，我們身體許 多疾病的根本原因常可追究於免疫系統的功能不健全。一般可分 為二種情況。第一種是抵抗力降低，身體的免疫細胞活力下降， 或生產的數量不足以抵抗入侵的細菌、病毒或黴菌，因此易被感 染流行病。傷風、感冒、肺炎、腸炎甚至於肝炎，愛滋病等，都 屬於這一類。第二種的免疫不全是免疫系統反應過度。這種情況 導因於入侵的物質並不是細菌而只是小花粉或是食物中大分子 蛋白質，免疫系統就釋出大量的抗體加以對抗，而攻勢就發生在 我們的細胞中，如此引起一連串的反應，也被稱為過敏症。而這 時候若果真有細菌、病毒或黴菌這類真的病原來侵犯時，免疫系 統已無餘力抵抗了。第三種情況是免疫細胞攻擊自體的正常細 胞’也就疋自體免疫疾病’例如：類風濕性關節炎、紅斑性狼瘡 或跑疹等等，這種免疫系統疾病起因於病患本身免疫辨識系統發 生問題，病患的免疫系統對於本身的某些物質會產生自體抗體 (autoantibody)，進而破壞身體組織造成病痛。 目前已知並非是只有自體免疫疾病才有自體抗體的存在，越 來越多的相關研究指出人類對癌症所發生的免疫反應，部分存在 著腫瘤自體抗原（autoantigen)與人類自體抗體的情形，因此對 於發現這些可以引起人體反應的腫瘤自體抗原在癌症檢測、診斷 或建立預後上有一定的方向和應用性，進而應用於疾病的免疫治 J281473 療上。 在美國專利 6631330、5137807、5830667、6264949、5985542 等五篇中，揭露使用生物標記分子診斷肝硬化（cirrhosis )、纖 維化（fibrosis)或自體免疫性肝炎（autoimmune hepatitis，AIH ); 而在美國專利4994374、5175084等兩篇中則使用生物標記分子 診斷肝癌（hepatcellular carcinoma);另外在美國專利6410724 則使用和肝癌相關的DNA引子（DNA primer)組成一檢測套組，但 這些生物標記分子多少都存在準確性不高或易被干擾的缺點。 在美國專利5891436及公開專利20030138860中，揭露了使 -用生物標記分子檢測人體血清中其自體抗體的存在，以做為硬化 (primary biliary cirrhosis )、癌症的判斷，這說明在癌症病人中 會有自體抗體（autoantibody)的產生，確立應用於篩選癌症相關 之生物標記分子（biomarker)的合理性。 自民國七Η--年以來，癌症即躍居臺灣地區十大死亡原因首 位。其中肝癌不分男性或女性都名列前茅，這對國人的健康造成 極大威脅，為有效篩檢罹患肝臟疾病的病患，期許早期治療降低 死亡率，發現高準確性及不被干擾的生物標記分子，以作為檢測 肝硬化及肝癌之檢測套組，便成為重要的課題。 【發明内容】 有鑑於習知技術之缺失，本發明係揭露肝臟疾病之生物標記 分子’由於確知其自體抗體的存在，故可設計成檢測套組，藉此 作為肝硬化或肝癌之診斷使用。 本發明之目的係關於一種用於檢測肝臟疾病之生物標記分 子’其係選自SEQ ID ΝΟ:1至SEQ ID ΝΟ:24所示之任一胺基酸 序列或其衍生物或其片段或其變異體或其組合物或其對應之自 體抗體。 前述肝臟疾病係為肝硬化或肝癌。 1281473 前述變異體係將生物標記分子之胺基酸序列中之胺基酸以 一個或多個胺基酸取代、刪除、插入及/或添加；變異體之胺基酸 序列與生物標記分子之胺基酸序列具有大於80%之序列同一性。 本發明之另一目的係關於一種肝臟疾病之檢測套組，包含一 組生物標記分子，其係選自SEQ ID ΝΟ:1至SEQ ID NO:24所示 之任一胺基酸序列或其衍生物或其片段或其變異體或其組合物。 前述肝臟疾病係為肝硬化或肝癌。 前述檢測套組係可進一步包含可辨識SEQ ID ΝΟ:1至SEQ ID ΝΟ·.24所示之任一胺基酸序列或其衍生物或其片段或其變異 體之抗體之二級抗體。 本發明之再一目的係關於一種肝臟疾病之筛檢方法，係包含 下列步驟··提供一檢體；利用SEQ ID ΝΟ:1至SEQ ID ΝΟ:24所 示之任一胺基酸序列或其衍生物或其片段或其變異體或其組合 物之生物標記分子辨識捕捉檢體中之自體抗體；及偵測自體抗 體。 前述肝臟疾病係為肝硬化或肝癌。 前述檢體係為全血或血清，其中以企清較佳。 前述生物標記分子係可為各種型式，包括，但不限於，檢測 套組、或先行固定於基材上，前述基材係為免痩分析盤或生物晶 片。 前述檢體係可進一步先行利用螢光標記進行標示。 前述篩檢方法係可增加一利用二級抗體辨識吸附自體抗體 之步驟。 前述二級抗體係經修飾具有特殊官能基可進行顯色反應、放 射性彳貞測或榮光彳貞測。 前述偵測自體抗體係利用酵素連結免疫吸附分析 (Enzyme-linked Immunosorbent Assay，ELIS A )、放射免疫分析 1281473 (Radioimmunoassa，RIA )或免疫螢光分析（Immunofluorescence ) 偵測二級抗體，或直接利用螢光掃描偵測被螢光標記標示之自體 抗體。 本發明之再一目的係關於一種肝臟疾病之檢測套組，包含一 組可辨識具有SEQ ID N0:1至SEQ ID NO:24所示之任一胺基酸 序列對應之抗體。 前述肝臟疾病係為肝硬化或肝癌。 本發明之再一目的係關於一種利用前述檢測套組篩檢肝臟 疾病之方法，係包含下列步驟：提供一檢體利用SEQ ID NO: 1 至SEQ ID NO:24所示之任一胺基酸序列對應之抗體辨識捕捉檢 體中之抗原；及偵測抗體與抗原之複合物。 前述肝臟疾病係為肝硬化或肝癌。 前述檢體係為全血或血清，其中以血清較佳。 本發明·之基礎係利用自體抗原篩選方法，先純化出正常人和 肝硬化、肝癌病人的抗體並分別固定於不同管柱中，再將人肝疾 病相關之細胞株（HepG2 C3A&SNU-387)的細胞萃取物依序通入 正常人及病人之管柱，以得到和肝硬化、肝癌等肝臟疾病相，關之 自體抗原，再以這些自體抗原做為檢測肝硬化及肝癌之生物標記 套組’搭配酵素連結免疫吸附分析法（Enzyme_Unked Immunosorbent Assay , ELISA )、放射免疫分析法 (Radioimmunoassay，RIA )或免疫螢光分析法 (Imnnm〇flU〇reSCence)等’藉檢測人體血清中是否有該自體抗 原（autoantigen)所相對應的自體抗體（aut〇amib〇dy)的存在判 斷是否罹患肝癌或肝硬化等肝臟疾病。由於這些生物標記分子皆 是以已存在之自體抗㈣選出來，所以在診斷上即可設計為檢測 套組藉生物心。己分子相對應之自體抗體存在與否，作為是否羅 患該疾病之麟’収直㈣檢抗原_難度要降低許多，可有 1281473 效提高準確度與靈敏度。 【實施方式】 本發明係關於利用自體抗原篩選方法，篩選出可用於檢測肝 硬化或肝癌等肝臟疾病之生物標記分子，自體抗原篩選方法如第 圖所示’包含以下步驟：首先取得正常人及病患之血清樣品， 分別利用可捕捉抗體之親和力管柱純化出血清樣品t所含之抗 體；接著將正常人之抗體與病患之抗體分別填充入管柱之中，以 製侍含正常人抗體之管柱及含病患抗體之管柱，抗體填充固定於 :柱之令之方法，係將由血清樣品純化出來的抗體與管柱中之化 學官能基形成化學鍵結固定於管柱中。然後取一樣品，此樣品可 為（癌）細胞株或者病理組織之萃取物；前述病患之血清樣品係 可為單一病患之血清樣品或複數病患之血清混合樣品。 將來自（癌）細胞株或病理組織萃取物的樣品通入正常人抗 體管柱時，可藉由正常人抗體的專一親和力將非專一性之抗原捕 捉而滯留於管柱之中，通過管柱的樣品將不含非專一性抗原，這 個程序其實可視為利用病患抗體管柱篩選檢體中自體抗原之樣 品前處理。經移除樣品中非專一性之抗原，樣品組成便剩下特有 之抗原，此時再將樣品通入含病患抗體之管柱中，藉由病患所具 有之自體抗體來進一步篩選疾病相關之自體抗原。由於已先藉由 正系人的血清抗體去除非專一性之抗原，所以利用病患之自體抗 體時選所得到的自體抗原會具有較高的專一性。 由病患抗體管柱中置換而出的自體抗原，係利用質譜技術進 行鑑定；前述之鑑定係將質譜訊號透過資料庫比對，以取得自體 抗原之資訊。 將人類肝臟疾病相關之細胞株利用上述方法進行自體抗原 之純化與鑑定，由於這些自體抗原係利用病患血清中之抗體篩選 出來，所以可利用這些自體抗原作為生物標記分子發展為檢測套 1281473 組，該檢測套組係選自前述自體抗原篩選方法篩選出之自體抗原 或其衍生物或其片段或其變異體或其組合物。藉篩選檢體中自體 抗體的存在，判斷是否罹患肝硬化或肝癌疾病。檢測套組中除生 物標記分子外，還可係可進一步包含可辨識該生物標記分子之自 體抗體之二級抗體，以利應用於相關檢測方法。 運用前述檢測套組篩檢之方法如第二圖所示：包含下列步 驟：首先提供一檢體；接著利用生物標記分子辨識捕捉檢體中之 自體抗體，然後偵測自體抗體。前述生物標記分子係選自自體抗 原篩選方法篩選出之自體抗原或其衍生物或其片段或其變異體 或其組合物。 前述檢體係為全血或血清，其中以血清較佳的選擇。 為有利於檢測的進行，前述生物標記分子可先行固定於基材 上，常見-之固定基材係為免疫分析盤或生物晶片。當生物標記分 子捕捉檢體中之自體抗體後，可利用二級抗體辨識吸附自體抗 體，這些二級抗體係經修飾具有特殊官能基，以進行顯色反應、 放射性偵測或螢光偵測。 利用二級抗體辨識吸附自體抗體後，即可加入特殊試劑進行 顯色反應，然後利用酵素連結免疫吸附分析（Enzyme-linked Immunosorbent Assay，ELISA)進行二級抗體存在之判斷，進而 得知自體抗體是否存在，或透過放射免疫分析 (Radioimmunoassay ， RIA )或免疫螢光分析

, I (Immunofluorescence )，同樣可以得知藉由二級抗體的反應判 斷自體抗體是否存在，以作為是否罹患肝癌或肝硬化之依據。 若不使用二級抗體，也可將檢體在與生物標記分子反應前， 先利用螢光標記（例如Cy3或Cy5)標示，然後透過生物標記分 子篩選自體抗體，由於自體抗體已先被螢光標記標示，所以在無 須使用二級抗體情況下即可進行螢光掃瞄分析。 10 1281473 除了偵測自體抗體的存在作為判斷，偵測抗原的存在亦可作 為是否罹患肝硬化或肝癌之依據，為達此目的，設計一種包含可 辨識具有自體抗原篩選方法篩選出之自體抗原之抗體的檢測套 組，亦可作為肝臟疾病檢測之用。 利用前述檢測套組篩檢肝癌及肝硬化之方法包含下列步 驟：提供一血清檢體；利用前述抗體辨識捕捉血清中之抗原；及 偵測抗體與抗原之複合物。 以下實施例係用於進一步了解本發明之優點，並非用於限制 本發明之申請專利範圍。 .實施例1·利用肝臟疾病病患血清中之自體抗體篩選自體抗原 由血清純化自體抗體： 首先取肝硬化或肝癌病患之血清，將血清用鍵結緩衝液 (binding buffer ·· 20 mM PBS，pH 7.0)以 1 : ί〇 的 t匕你J 稀釋後， 以0.45 //m孔徑的濾膜過濾，避免後續層析步驟造成管柱堵塞； 接著以10倍管柱體積的鍵結緩衝液，在1 ml/min的流速下清洗 Protein G親和力管柱，之後將前述經濾、膜過濾之血清樣本以0.2 ml/min的流速通入Protein G親和力管柱中，使抗體得以藉由與 管柱之親和力滯留於管柱中；再取5-10倍管柱體積的鍵結緩衝液 以1 ml/miri的流速清洗Protein G親和力管柱，移除血清樣品中 不與管柱發生親和力鍵結之物質，然後取2-5倍管柱體積之沖提 緩衝液（elution buffer : 0.1 M Glycine-HCl，pH 2.7 )以 1 ml/min 的流速將抗體自管柱中沖提出並收集，收集抗體之試管中需預先 加入60-200/z 1之Tris-HCl溶液（1 Μ，pH 9.0)，最後將樣品置 換於耦合緩衝液（coupling buffer : 0.2 M NaHC03，0·5 M NaCl， ρΗ8·3)中，即完成血清中自體抗體（IgG)之純化。 由於本發明需要填充有正常人IgG及病患IgG之管柱各一， 故需取正常人及病患血清分別進行前述純化步驟。 128147¾ 製備含有自體抗體之管柱： 取NHS-activated管柱一只，移除上端蓋子後滴一滴酸化溶 液（acidification solution : 1 mM HC1 (需冰浴））以避免氣泡生 成。接著將此管柱上端與注射器（syringe)或泵浦（pump)連接 後移除下端扭轉處，再以2倍管柱體積之酸化溶液將管柱中之異 丙醇清洗出來，重複此清洗步驟3次後，將含有自體抗體之樣品 注入管柱中。前述之含有自體抗體之樣品係由Protein-G親和力 管柱純化血清得到的含有自體抗體之輪合緩衝液，經配製後成為 總體積為1倍管柱體積，濃度為0.5-10 mg/rn卜將前述含有自體 抗體之樣品通入管柱後則將管柱密封，在25°C中反應15-30分 鐘’或者在4°C _反應4小時，使抗體得以與管柱產生化學鍵結 固定於管柱之中。 自體抗體與管柱完成鍵結反應後，先利用2倍管柱體積之封

阻緩衝液（blocking buffer : 0·5 M ethanolamine，0.5 M NaCl，pH 8·3 )沖提管柱，重複此步驟3次，再利用2倍管柱體積之清洗緩 衝液（washing buffer : 0· 1 M acetate，0.5 M NaCl，pH 4 )清洗管 柱’同樣重複3次，接著再換回封阻緩衝液以2倍管柱體積沖提 管柱3次後，讓管柱反應15-30分鐘，使管柱中未鍵結自體抗體 之官能基被封阻而失去活性，待封阻反應完成後以2倍管柱體積 之清洗緩衝液清洗管柱，重複3次，接著以2倍管柱體積之封阻 緩衝液沖提管柱，重複3次，確保所有未鍵結自體抗體之官能基 都能被封阻，再以2倍管柱體積之清洗緩衝液清洗管柱，重複3 次後，最後以2-5倍管柱體積之一般中性pH緩衝液沖提管柱後， 完成含有自體抗體之管柱的製備。 由肝贜疾病相關之細胞株萃取物中純化鑑定自體抗原： 首先將移除培養基之HepG2 C3A cells 2.68 mg以冰浴之Tris saline 溶液（50 mM Tris pH 7·5，150 mM NaCl，1.5 mM PMSF， 12 1281473

Phosphatase inhibitors)清洗兩次後，加入 Triton Extraction 溶液 lml ( 15 Mm Tris pH 7·5，120 mM NaCl，25 mM KC1，2 mM EGTA，0.1 mM DTT，0.5% Triton X-100，10 # g/ml leupeptin， 0.5 Mm PMSF，Phosphatase inhibitors)，置於 4°C、30 分鐘，此 時細胞開始分解釋出蛋白質，然後利用桌上型離心機於4°C以 14,000 rpm的轉速離心15分鐘，移除固態不溶之細胞結構，收集 上層液進行後續之免疫親和層析。 經收集得到的細胞萃取液與鍵結緩衝液（binding buffer )以 1:10的比例稀釋後，利用0.45 # m孔徑的濾膜過濾，以避免後續 通入管柱後造成管柱的阻塞，而在將樣品注入具有抗體（IgG) 的管柱前，需先以10倍管柱體積的鍵結緩衝液，在1 ml/min的 流速下清洗正常人及病患之抗體管柱，之後將前述經濾膜過濾之 細胞萃取液以0.2 ml/min的流速通入正常人抗體管柱中，再取 5-10倍管柱體積的鍵結緩衝液以1 ml/min的流速沖提正常人抗體 管柱，此時細胞萃取液中所含有之正常人抗體具辨識捕捉的抗原 會滯留其中，此步驟之目的在捕捉移除細胞株中非專一性之抗 原，所以經管柱層析後之細胞萃取液中將不含非專一性之抗原； 將此細胞萃取液收集導入病患抗體管柱，取5-10倍管柱體積的鍵 結緩衝液以1 ml/min的流速沖提病患抗體管柱，此時細胞萃取液 中所具有之自體抗原將被管柱中病患之自體抗體所捕捉而滯留 於管柱之中，當細胞萃取液通過正常人抗體管柱時，一般正常人 抗體可辨識捕捉之抗原將被滯留於管柱中，而將不含前述正常人 抗體可辨識捕捉之抗原之細胞萃取液導入病患抗體管柱時，只有 病患抗體管柱可辨識捕捉之抗原被滯留於管柱之中，這些滞留於 病患抗體管柱中之抗原，係藉由2-5倍管柱體積之沖提緩衝液 (elution buffer )以1 mi/min的流速將自體抗原由病患抗體管柱 中沖提出並收集，接著使用胰蛋白酵素（Trypsin)進行蛋白質水 13 1281473 * 解反應，將被水解之自體抗原之胜肽以質譜技術進行分析，所得 之圖譜可藉由資料庫比對獲知蛋白質之資訊。 利用肝硬化或肝癌病患血清中之自體抗體篩選肝臟疾病相 關細胞株，可得下列之自體抗原： 1. Nucleoside diphosphate kinase ( gi| 1421609 j SEQ ID NO. 1 ) 2. NM23 protein ( gi|35068，SEQ ID N0.2 ) 3. ATP synthase beta chain，mitochondrial [precursor] (gi|28940，SEQIDN0.3) 4. 14-3-3 zeta protein(tyrosine 3/tryptophan 5 -monooxygenase activation protein) ( gi|4507953，SEQ ID NO.4) 5. 14-3-3 epsilon protein(tyrosine 3/tryptophan 5 -monooxygenase activation protein) ( gi|4507953 ^ SEQ ID NO.5) 6. Protein disulfide isomerase-related protein 5 ( gi| 1710248 » SEQ ID NO.6) 7. unnamed protein product ( gi|21750187，SEQ ID N0.7 ) 8. Tropomyosin alpha 3 ( gi|37403 ? SEQ ID NO.8 ) 9. Trypomyosin alpha 4 ( gi|10435300，SEQ ID NO.9) 10. Galreticulin precursor ( gi|4757900 ? SEQ ID NO.10) 11. Human pre-mRNA splicing factor SF2p32 ( gi|338043 5 SEQ ID NO.11 ) 12. Tumor necrosis factor type 1 receptor associated protein TRAP-1 (gi|1082886, SEQ ID NO.12) 13. Tumor rejection antigen( gp96 ) 1 ； glucose regulated protein (gi|4507677，SEQIDNO.13) 14. Heat shock protein 90-beta ( gi|72222 9 SEQ ID NO.14) 14 1281473 15. Heat shock 90-alpha ( gi|123678 ^ SEQ ID NO.15) 16. Heat shock 60kDa protein 1 ( gi|3 1542947 ^ SEQ ID NO.16) 17. HMG-1 (gi|968888, SEQIDNO.17) 18. KIAA0144 gene product ( NICE-4 protein) ( gi| 13 1 1 1995， SEQ ID NO.18) 19. Valosin-containing protein( p97 ) ； transitional endoplasmic reciculum ATPase ( gi|6005942，SEQ ID ΝΟ·19) 20:Glyceraldehyde 3-phosphate dehydrogenase，liver (gi|30157565，SEQIDNO.20) 21.Cytokeratin ( gi|1419564，SEQ ID ΝΟ·21 ) — 22.IGF-II mRNA-binding protein 1 ( gi|4191608，SEQ ID NO.22) 23. NADPH： quinone reductase ( gi| 13236495 ^ SEQ ID NO.23 ) 24. Crystal Structure Of The Human Co-Chaperone P23( hsp-90 co-chaperone) ( gi|9257073 ^ SEQ ID NO.24) 上列由肝臟疾病相關之細胞株篩選之自體抗原如表一所 示，其中左側為蛋白質之GI編號（gi number )及名稱，右側為 利用肝硬化或肝癌病患之血清在肝臟疾病相關細胞株中可被篩 選出來的自體抗原，由表中顯示，這些自體抗原並不侷限於單一 之肝臟疾病中才會被篩選出來，利用不同來源血清中之自體抗體 針對不同細胞株也會重複篩選出來，顯示這些抗原與肝臟疾病之 相關性極為密切。表一中部分蛋白質具有兩個GI編號，其係肇 因於蛋白質及其變異體在質譜鑑定時具有近似之判別結果。 15 1281473 噼^|| 孤 W 钳蹬泰驾4w^^^^Mt-ffi,u< 肝癌血清 VS. SNU-387 • • • 肝硬化血清 VS. SNU-387 • • • • 肝癌血清 VS. HepG2 C3A • • • • • • • • ·· • • 名 G 念-3 货 〇, • • • 蛋白質名稱 Nucleoside Diphosphate Kinase( = NM23 protein) ATP synthase beta chain, mitochondrial [Precursor] 14-3-3 protein Protein disulfide isomerase-related protein 5 Gi|21750187 Unname protein product (RNA rec mot.) Tropomyosin Calreticulin precursor Human pre-mRNA splicing factor SF2p32, complete sequence Tumor necrosis factor type 1 receptor associated protein TRAP-1 Tumor rejection antigen (gp96) 1; glucose regulated protein 1 Heat shock protein 90 Heat shock 60kDa protein 1 (chaperonin); mitochondrial matrix protein PI HMG-1 (high-mobility group-1) KIAA0144 gene product (NICE-4 protein ) Valosin-containing protein(p97); transitional endoplasmic reticulum ATPase Glyceraldehyde 3-phosphate dehydrogenase, liver Cytokeratin IGF-II mRNA-binding protein 1 NADPH:quinone reductase Crystal Structure Of The Human Co-Chaperone P23 (hsp-90 co-chaperone) GI number 1421609 28940 CN (N s 00 rn Os 1710248 21750187 37403,10435300 4757900 338043 1082886 4507677 72222,123678 31542947 968888 13111995 6005942 30157565 1419564 4191608 13236495 9257073 1281473 實施例2·鑑定利用自體抗原篩選方法篩選出來之自體抗原 之可利用性 為進一步證實實施例1 ·篩選出之24個自體抗原之·可利 用性，需進一步使用免疫分析方法（ELISA、RIA、免疫螢 光法等）搭配上述24個生物標記分子，分別對正常人、肝 硬化病患及肝癌病患之血清樣品做進一步檢測，其檢測方法 依循第二圖，首先提供一檢體，然後利用SEQ ID N0:1至 SEQ ID N〇:24所示之任一胺基酸序列或其衍生物或其片段 或其組合物之生物標記分子辨識捕捉檢體中之自體抗體，最 後偵測自體抗體。 以酵素連結免疫吸附分析 （Enzyme-linked Immunosorbent Assay，ELIS A )為例，其詳細檢測步驟如下： 首先將生物標記分子以彼覆緩衝液（ coating buffer·，可有三 楂選擇為 a· 50mM Na2HC03，pH=9.6 或 b. 20mM Tris-Ηα， pH=8.5 或.c. l〇mM PBS，pH=7.4)稀釋成濃度 0.5〜10/z g/ml，彼覆緩衝液係依據生物標記分子之pi值選擇，以大 於pi值1〜2 pH值之緩衝液為較佳。在ELISA plate中加入 100/zl/well之生物標記分子溶液，置於4°C中反應隔夜進行 固定化。 i

接著移除未固定化之生物標記分子，以PBST buffer*清 洗兩次（PBST buffer : PBS buffer+0.05% Tween-2.0)，然 後加入200 # 1/well的封阻緩衝液（blocking buffer :可有三 種選擇 a· Gelatin-NET : 0.5% Gelatin，0.15M NaCl，5mM EDTA · 2Na，0.05% Tween-20，50mM Tris base 或 b. 1% BSA_PBS，ρΗ=7·4 或 5% Non-fat milk-PBS，pH=7.4)在室 溫下反應最少2小時進行封阻反應；反應完成後利用PBST 17 1281473 緩衝液清洗3次，然後加入100 // l/well待反應之血清溶液 (血清溶液係使用封阻緩衝液稀釋1000倍），此時血清中 之自體抗體將與被固定化之生物標記分子進行辨識吸附，此 反應於室溫下至少進行2小時後，利用PBST緩衝液清洗4 次，再加入100 V Ι/well之二級抗體（二級抗體係利用封阻 緩衝液稀釋5000倍），此時二級抗體將會與自體抗體進行 辨識吸附，此反應於室溫下至少進行1小時候，以PBST緩 衝液清洗5次，接著加入100/z Ι/well之TMB進行30分鐘 顯色反應，然後加入ΙΟΟβΙ/well之0·5Μ H2S04後，偵測 450nm之吸收度。 為確認自體抗體的表現量確實可作為肝硬化及/或肝癌 之檢測，可分別利用ELISA分析各自體抗原篩選正常人、 肝硬化病患及肝癌病患血清中之自體抗體之吸光值數據 後，以生物統計學進行分析以GADPH、NADPH、HMG-1、 NM23 及 Cytokeratin 等五個蛋白質為例，利用 Wilcoxon-Mann-Whitney進行檢定，在95%之信賴水準下所 得結果如下表： GADPH NADPH HMG-1 NM23 Cytokeratin 正常人 VS. 肝硬化病患 P=0.001 ρ=0·001 p=0.00006 p=0.0001 p=0.00 1 , Λ 1 正常人 VS. 肝癌病患 P = 0.017 p=0.016 p=0.0 1 5 p=0.002 p=0.0 16 肝硬化病患 VS. 肝癌病患 p>0.05 p>0.05 p>0.05 p>0.05 p>0.05 正常人：N=10 、肝硬化病患·· N=15、肝癌病患：N=21 (p<0.05假設成立） 18 1281473 假設各生物標記分子之自體抗體於正常人、肝硬化病患 及肝癌病患中表現量具差異性，則由表中顯示這樣的假設在 正常人與肝硬化病患的比較，以及正常人與肝癌病患之比較 之假設成立，意即各生物標記分子之自體抗體之表現量在正 常人與肝硬化病患及肝癌病患中之差異，具有生物統計上的 意義存在。 經由生物統計可知蛋白質GADPH之自體抗體在正常人 與肝硬化病患中之表現量差異達8.375倍，在正常人與肝癌 病患中之表現量差異達4,86倍；蛋白質HMG-丨之自體抗體 在正常人與肝硬化病患中之表現量差異達74倍；蛋白質 NM23之自體抗體在正常人與肝硬化病患中之表現量達24 倍，在正常人與肝癌病患中之表現量達8.545倍。這些結果 證實在肝硬化及肝癌病人的血清中，此24個自體抗原之相 關自體抗體（autoantibodies)的表現量高於正常人，因此將上 述24個自體抗原搭配免疫分析方法組合成一套檢測套組， 藉此篩選檢體中自體抗體表現量，確實可以作為肝硬化及肝 癌之檢測用。 雖然本發明已以較佳實施例揭露如上，然其並非用以限 定本發明，任何熟悉此技藝者，在不脫離本發明之精神和範 圍内，當可作各種之更動與潤飾，因此，本發明之保護範圍， 當視後附之申請專利範圍所界定者為準。 19 1281473 【圖式簡單說明】 第一圖係為利用自體抗原篩選方法之流程圖 第二圖係為利用生物標記分子篩選自體抗體之流程圖。 20 1281473 ··, 序列表 <110>財團法人工業技術研究院 < 120〉肝臟疾病之生物標記分子及其檢測方法 <160〉 24 <210> 1 <211> 151 <212> PRT <213〉人屬、人種 <400〉 1

Ala Asn Leu Glu Arg Thr Phe lie Ala lie Lys Pro Asp Gly Val Gin 1 5 10 15

Arg Gly Leu Val Gly Glu He He Lys Arg Phe Glu Gin Lys Gly Phe 20 25 30

Arg Leu Val Ala Met Lys Phe Leu Arg Ala Ser Glu Glu His Leu Lys 35 40 45

Gin His Tyr lie Asp Leu Lys Asp Arg Pro Phe Phe Pro Gly Leu Val 50 55 60

Lys Tyr Met Asn Ser Gly Pro Val Val Ala Met Val Trp Glu Gly Leu 65 70 75 80

Asn Val Val Lys Thr Gly Arg Val Met Leu Gly Glu Thr Asn Pro Ala 85 90 95

Asp Ser Lys Pro Gly Thr lie Arg Gly Asp Phe Cys lie Gin Val Gly 100 105 110 '

Arg Asn He He His Gly Ser Asp Ser Val Lys Ser Ala Glu Lys Glu 115 120 125 lie Ser Leu Trp Phe Lys Pro Glu Glu Leu Val Asp Tyr Lys Ser Cys 130 135 140

Ala His Asp Trp Val Tyr Glu 145 150 <210〉 2 <211> 180 <212> PRT 1281473 、 <213〉人屬、人種 <400> 2

Cys Cys Glu Pro Arg Gly Ser Arg Ala Arg Phe Gly Cys Trp Arg Leu 1 1 5 Γ :10 ; 15、

Gin Pro Glu Phe Lys Pro Lys Gin Leu Glu Gly Thr Met Ala Asn Cys 20 25 30

Glu Arg Thr Phe He Ala He Lys Pro Asp Gly Val Gin Arg Gly Leu 35 40 45

Val Gly Glu lie lie Lys Arg Phe Glu Gin Lys Gly Phe Arg Leu Val 50 55 60

Gly Leu Lys Phe Met Gin Ala Ser Glu Asp Leu Leu Lys Glu His Tyr 65 70 75 80

Val Asp Leu Lys Asp Arg Pro Phe Phe Ala Gly Leu Val Lys Tyr Met 85 90 95

His Ser Gly Pro Val Val Ala Met Val Trp Glu Gly Leu Asn Val Val 100 105 110

Lys Thr Gly Arg Val Met Leu Gly Glu Thr Asn Pro Ala Asp Ser Lys 115 120 125

Pro Gly Thr lie Arg Gly Asp Phe Cys lie Gin Val Gly Arg Asn lie 130 135 140

He His Gly Ser Asp Ser Val Glu Ser Ala Glu Lys Glu lie Gly Leu 145 150 155 160

Trp Phe His Pro Glu Glu Leu Val Asp Tyr Thr Ser Cys Ala Gin Asn 165 170 175

Trp lie Tyr Glu 180 <210〉 3 <211〉 539 <212> PRT <213>人屬、人種 <400〉 3

Met Thr Ser Leu Trp Gly Lys Gly Thr Gly Cys Lys Leu Phe Lys Phe 15 10 15

Arg Val Ala Ala Ala Pro Ala Ser Gly Ala Leu Arg Arg Leu Thr Pro 20 25 30 1281473

Ser Ala Ser Leu Pro Pro Ala Gin Leu Leu Leu Arg Ala Val Arg Arg 35 40 45

Arg Ser His Pro Val Arg Asp Tyr Ala Ala Gin Thr Ser Pro Ser Pro 50 55 60

Lys Ala Gly Ala Ala Thr Gly Arg lie Val Ala Val lie Gly Ala Val 65 70 75 80

Val Asp Val Gin Phe Asp Glu Gly Leu Pro Pro lie Leu Asn Ala Leu 85 90 95

Glu Val Gin Gly Arg Glu Thr Arg Leu Val Leu Glu Val Ala Gin His 100 105 110

Leu Gly Glu Ser Thr Val Arg Thr lie Ala Met Asp Gly Thr Glu Gly 115 120 125

Leu Val Arg Gly Gin Lys Val Leu Asp Ser Gly Ala Pro He Lys lie 130 135 140

Pro Val Gly Pro Glu Thr Leu Gly Arg lie Met Asn Val He Gly Glu 145 150 155 160

Pro lie Asp Glu Arg Gly Pro He Lys Thr Lys Gin Phe Ala Pro He 165 170 175

His Ala Glu Ala Pro Glu Phe Met Glu Met Ser Val Glu Gin Glu He 180 185 190

Leu Val Thr Gly He Lys Val Val Asp Leu Leu Ala Pro Tyr Ala Lys 195 200 205

Gly Gly Lys lie Gly Leu Phe Gly Gly Ala Gly Val Gly Lys Thr Val 210 215 220

Leu He Met Glu Leu lie Asn Asn Val Ala Lys Ala His Gly Gly Tyr 225 230 235 240

Ser Val Phe Ala Gly Val Gly Glu Arg Thr Arg Glu Gly Asn Asp Leu 245 250 255

Tyr His Glu Met He Glu Ser Gly Val He Asn Leu Lys Asp Ala Thr 260 265 270

Ser Lys Val Ala Leu Val Tyr Gly Gin Met Asn Gin Pro Pro Gly Ala 275 280 285

Arg Ala Arg Val Ala Leu Thr Gly Leu Thr Val Ala Glu Tyr Phe Arg 290 295 300

Asp Gin Glu Gly Gin Asp Val Leu Leu Phe lie Asp Asn lie Phe Arg 305 310 315 320

Phe Thr Gin Ala Gly Ser Glu Val Ser Ala Leu Leu Gly Arg He Pro 325 330 335 1281473

Ser Ala Val Gly Tyr Gin Pro Thr Leu Ala Thr Asp Met Gly Thr Met 340 345 350

Gin Glu Arg lie Thr Thr Thr Lys Lys Gly Ser lie Thr Ser Val Gin 355 360 365

Ala He Tyr Val Pro Ala Asp Asp Leu Thr Asp Pro Ala Pro Ala Thr 370 375 380

Thr Phe Ala His Leu Asp Ala Thr Thr Val Leu Ser Arg Ala He Ala 385 390 395 400

Glu Leu Gly lie Tyr Pro Ala Val Asp Pro Leu Asp Ser Thr Ser Arg 405 410 415

He Met Asp Pro Asn He Val Gly Ser Glu His Tyr Asp Val Ala Arg 420 425 430

Gly Val Gin Lys lie Leu Gin Asp Tyr Lys Ser Leu Gin Asp lie lie 435 440 445

Ala lie Leu Gly Met Asp Glu Leu Ser Glu Glu Asp Lys Leu Thr Val 450 455 460

Ser Arg Ala Arg Lys lie Gin Arg Phe Leu Ser Gin Pro Phe Gin Val 465 470 475 480

Ala Glu Val Phe Thr Gly His Met Gly Lys Leu Val Pro Leu Lys Glu 485 490 495

Thr lie Lys Gly Phe Gin Gin lie Leu Ala Gly Glu Tyr Asp His Leu 500 505 510

Pro Glu Gin Ala Phe Tyr Met Val Gly Pro lie Glu Glu Ala Val Ala 515 520 525

Lys Ala Asp Lys Leu Ala Glu Glu His Ser Ser 530 535 <210> 4 <211> 245 <212> PRT <213>人屬、人種 <400〉 4

Met Asp Lys Asn Glu Leu Val Gin Lys Ala Lys Leu Ala Glu Gin Ala 15 10 15

Glu Arg Tyr Asp Asp Met Ala Ala Cys Met Lys Ser Val Thr Glu Gin 20 25 30

Gly Ala Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 1281473 35 40 45

Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Val Ser Ser 50 55 60 lie Glu Gin Lys Thr Glu Gly Ala Glu Lys Lys Gin Gin Met Ala Arg 65 70 75 80

Glu Tyr Arg Glu Lys lie Glu Thr Glu Leu Arg Asp lie Cys Asn Asp 85 90 95

Val Leu Ser Leu Leu Glu Lys Phe Leu He Pro Asn Ala Ser Gin Ala 100 105 110

Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr Arg Tyr 115 120 125

Leu Ala Glu Val Ala Ala Gly Asp Asp Lys Lys Gly lie Val Asp Gin 130 135 140

Ser Gin Gin Ala Tyr Gin Glu Ala Phe Glu lie Ser Lys Lys Glu Met 145 150 155 160

Gin Pro Thr His Pro lie Arg Leu Gly Leu Ala Leu Asn Phe Ser Val 165 170 175

Phe Tyr Tyr Glu He Leu Asn Ser Pro Glu Lys Ala Cys Ser Leu Ala 180 185 190

Lys Thr Ala Phe Asp Glu Ala He Ala Glu Leu Asp Thr Leu Ser Glu 195 200 205

Glu Ser Tyr Lys Asp Ser Thr Leu He Met Gin Leu Leu Arg Asp Asn 210 215 220

Leu Thr Leu Trp Thr Ser Asp Thr Gin Gly Asp Glu Ala Glu Ala Gly 225 230 235 240

Glu Gly Gly Glu Asn 245 <210> 5 <211> 255 <212> PRT ' <213〉人屬、人種 <400〉 5

Met Asp Asp Arg Glu Asp Leu Val Tyr Gin Ala Lys Leu Ala Glu Gin 15 10 15

Ala Glu Arg Tyr Asp Glu Met Val Glu Ser Met Lys Lys Val Ala Gly 20 25 30 5 1281473

Met Asp Val Glu Leu Thr Val Glu Glu Arg Asn Leu Leu Ser Val Ala 35 40 45

Tyr Lys Asn Val lie Gly Ala Arg Arg Ala Ser Trp Arg He lie Ser 50 55 60

Ser lie Glu Gin Lys Glu Glu Asn Lys Gly Gly Glu Asp Lys Leu Lys 65 70 75 80

Met He Arg Glu Tyr Arg Gin Met Val Glu Thr Glu Leu Lys Leu lie 85 90 95

Cys Cys Asp lie Leu Asp Val Leu Asp Lys His Leu lie Pro Ala Ala 100 105 110

Asn Thr Gly Glu Ser Lys Val Phe Tyr Tyr Lys Met Lys Gly Asp Tyr 115 120 125

His Arg Tyr Leu Ala Glu Phe Ala Thr Gly Asn Asp Arg Lys Glu Ala 130 135 140

Ala Glu Asn Ser Leu Val Ala Tyr Lys Ala Ala Ser Asp He Ala Met 145 150 155 160

Thr Glu Leu Pro Pro Thr His Pro He Arg Leu Gly Leu Ala Leu Asn 165 170 175

Phe Ser Val Phe Tyr Tyr Glu lie Leu Asn Ser Pro Asp Arg Ala Cys 180 185 190

Arg Leu Ala Lys Ala Ala Phe Asp Asp Ala He Ala Glu Leu Asp Thr 195 200 205

Leu Ser Glu Glu Ser Tyr Lys Asp Ser Thr Leu lie Met Gin Leu Leu 210 215 220

Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Met Gin Gly Asp Gly Glu 225 230 235 240

Glu Gin Asn Lys Glu Ala Leu Gin Asp Val Glu Asp Glu Asn Gin 245 250 255 <210〉 6 <211〉 421 <212〉 PRT <213〉人屬、人種 <400〉 6

Leu Tyr Ser Ser Ser Asp Asp Val lie Glu Leu Thr Pro Ser Asn Phe 15 10 15

Asn Arg Glu Val He Gin Ser Asp Ser Leu Trp Leu Val Glu Phe Tyr 1281473 20 25 30

Ala Pro Trp Cys Gly His Cys Gin Arg Leu Thr Pro Glu Trp Lys Lys 35 40 45

Ala Ala Thr Ala Leu Lys Asp Val Val Lys Val Gly Ala Val Asp Ala 50 55 60

Asp Lys His His Ser Leu Gly Gly Gin Tyr Gly Val Gin Gly Phe Pro 65 70 75 80

Thr lie Lys lie Phe Gly Ser Asn Lys Asn Arg Pro Glu Asp Tyr Gin 85 90 95

Gly Gly Arg Thr Gly Glu Ala He Val Asp Ala Ala Leu Ser Ala Leu 100 105 110

Arg Gin Leu Val Lys Asp Arg Leu Gly Gly Arg Ser Gly Gly Tyr Ser 115 120 125

Ser Gly Lys Gin Gly Arg Ser Asp Ser Ser Ser Lys Lys Asp Val lie 130 135 140

Glu Leu Thr Asp Asp Ser Phe Asp Lys Asn Val Leu Asp Ser Glu Asp 145 150 155 160

Val Trp Met Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Asn 165 170 175

Leu Glu Pro Glu Trp Ala Ala Ala Ala Ser Glu Val Lys Glu Gin Thr 180 185 190

Lys Gly Arg Val Lys Leu Ala Ala Val Asp Ala Thr Val Asn Gin Val 195 200 205

Leu Ala Ser Arg Tyr Gly He Arg Gly Phe Pro Thr lie Lys He Phe 210 215 220

Gin Lys Gly Glu Ser Pro Val Asp Tyr Asp Gly Gly Arg Thr Arg Ser 225 230 235 240

Asp lie Val Ser Arg Ala Leu Asp Leu Phe Ser Asp Asn Ala Pro Pro 245 250 255

Pro Glu Leu Leu Glu He He Asn Glu Asp lie Ala Lys Arg Thr Cys 260 265 270

Glu Glu His Gin Leu Cys Val Val Ala Val Leu Pro His lie Leu Asp 275 280 285

Thr Gly Ala Ala Gly Arg Asn Ser Tyr Leu Glu Val Leu Leu Lys Leu 290 295 300

Ala Asp Lys Tyr Lys Lys Lys Met Trp Gly Trp Leu Trp Thr Glu Ala 305 310 315 320

Gly Ala Gin Ser Glu Leu Glu Thr Ala Leu Gly lie Gly Gly Phe Gly 1281473 325 330 335

Tyr Pro Ala Met Ala Ala lie Asn Ala Arg Lys Met Lys Phe Ala Leu 340 345 350

Leu Lys Gly Ser Phe Ser Glu Gin Gly lie Asn Glu Phe Leu Arg Glu 355 360 365

Leu Ser Phe Gly Arg Gly Ser Thr Ala Pro Val Gly Gly Gly Ala Phe 370 375 380

Pro Thr He Val Glu Arg Glu Pro Trp Asp Gly Arg Asp Gly Glu Leu 385 390 395 400

Pro Val Glu Asp Asp He Asp Leu Ser Asp Val Glu Leu Asp Asp Leu 405 410 415

Gly Lys Asp Glu Leu 420 <210> 7 <211〉 687 <212> PRT <213〉人屬、人種 <400> 7

Met Val Lys Leu Ala Lys Ala Gly Lys Asn Gin Gly Asp Pro Lys Lys 1 5 10 15

Met Ala Pro Pro Pro Lys Glu Val Glu Glu Asp Ser Glu Asp Glu Glu 20 25 30

Met Ser Glu Asp Glu Glu Asp Asp Ser Ser Gly Glu Glu Val Val He 35 40 45

Pro Gin Lys Lys Gly Lys Lys Ala Ala Ala Thr Ser Ala Lys Lys Val 50 55 60

Val Val Ser Pro Thr Lys Lys Val Ala Val Ala Thr Pro Ala Lys Ala 65 70 75 80

Val Thr Thr Pro Gly Lys Lys Gly Ala Thr Pro Gly Lys Ala Leu Val 85 90 95

Ala Thr Pro Gly Lys Lys Gly Ala Ala lie Pro Ala Lys Gly Ala Lys 100 105 110

Asn Gly Lys Asn Ala Lys Lys Glu Asp Ser Asp Glu Glu Glu Asp Asp 115 120 125

Asp Ser Glu Glu Asp Glu Glu Asp Asp Glu Asp Glu Asp Glu Asp Glu 130 135 140 8 1281473

Asp Glu lie Glu Pro Ala Ala Met Lys Ala Ala Ala Ala Ala Pro Ala 145 150 155 160

Ser Glu Asp Glu Asp Asp Glu Asp Asp Glu Asp Asp Glu Asp Asp Asp 165 170 175

Asp Asp Glu Glu Asp Asp Ser Glu Glu Glu Ala Met Glu Thr Thr Pro 180 185 190

Ala Lys Gly Lys Lys Ala Ala Lys Val Val Pro Val Lys Ala Lys Asn 195 200 205

Val Ala Glu Asp Glu Asp Glu Glu Glu Asp Asp Glu Asp Glu Asp Asp 210 215 220

Asp Asp Asp Glu Asp Asp Glu Asp Asp Asp Asp Glu Asp Asp Glu Glu 225 230 235 240

Glu Glu Glu Glu Glu Glu Glu Glu Pro Val Lys Glu Ala Pro Gly Lys 245 250 255

Arg Lys Lys Glu Met Ala Lys Gin Lys Ala Ala Pro Glu Ala Lys Lys 260 265 270

Gin Lys Val Glu Gly Thr Glu Pro Thr Thr Ala Phe Asn Leu Phe Val 275 280 285

Gly Asn Leu Asn Phe Asn Lys Ser Ala Pro Glu Leu Lys Thr Gly lie 290 295 300

Ser Asp Val Phe Ala Lys Asn Asp Leu Ala Val Val Asp Val Arg lie 305 310 315 . 320

Gly Met Thr Arg Lys Phe Gly Tyr Val Asp Phe Glu Ser Ala Glu Asp 325 330 335

Leu Glu Lys Ala Leu Glu Leu Thr Gly Leu Lys Val Phe Gly Asn Glu 340 345 350

He Lys Leu Glu Lys Pro Lys Gly Lys Asp Ser Lys Lys Glu Arg Asp 355 360 365

Ala Arg Thr Leu Leu Ala Lys Asn Leu Pro Tyr Lys Val Thr Gin Asp 370 375 380

Glu Leu Lys Glu Val Phe Glu Asp Ala Ala Glu He Arg Leu Val Ser 385 390 395 400

Lys Asp Gly Lys Ser Lys Gly He Ala Tyr He Glu Phe Lys Thr Glu 405 410 415

Ala Asp Ala Glu Lys Thr Phe Glu Glu Lys Gin Gly Thr Glu He Asp 420 425 430

Gly Arg Ser lie Ser Leu Tyr Tyr Thr Gly Glu Lys Gly Gin Asn Gin 435 440 445 1281473

Asp Tyr Arg Gly Gly Lys Asn Ser Thr Trp Ser Gly Glu Ser Lys Thr 450 455 460

Leu Val Leu Ser Asn Leu Ser Tyr Ser Ala Thr Glu Glu Thr Leu Gin 465 470 475 480

Glu Val Phe Glu Lys Ala Thr Phe lie Lys Val Pro Gin Asn Gin Asn 485 490 495

Gly Lys Ser Lys Gly Tyr Ala Phe lie Glu Phe Ala Ser Phe Glu Asp 500 505 510

Ala Lys Glu Ala Leu Asn Ser Cys Asn Lys Arg Glu lie Gly Gly Arg 515 520 525

Ala lie Arg Leu Glu Leu Gin Gly Pro Arg Gly Ser Pro Asn Ala Arg 530 535 540

Ser Gin Pro Ser Lys Thr Leu Phe Val Lys Gly Leu Ser Glu Asp Thr 545 550 555 560

Thr Glu Glu Thr Leu Lys Glu Ser Phe Asp Gly Ser Val Arg Ala Arg 565 570 575 lie Val Thr Asp Arg Glu Thr Gly Ser Ser Lys Gly Phe Gly Phe Val 580 585 590

Asp Phe Asn Ser Glu Glu Asp Ala Lys Ala Ala Lys Glu Ala Met Glu 595 600 605

Asp Gly Glu He Asp Gly Asn Lys Val Thr Leu Asp Trp Ala Lys Pro 610 615 620

Lys Gly Glu Gly Gly Phe Gly Gly Arg Gly Gly Gly Arg Gly Gly Phe 625 630 635 640

Gly Gly Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly Phe Gly Gly Arg 645 650 655

Gly Arg Gly Gly Phe Gly Gly Arg Gly Gly Phe Arg Gly Gly Arg Gly 660 665 670

Gly Gly Gly Asp His Lys Pro Gin Gly Lys Lys Thr Lys Phe Glu 675 680 085 <210> 8 <211〉 641 <212〉 PRT <213〉人屬、人種 <400> 8

Met Ala Gly lie Thr Thr lie Glu Ala Val Lys Arg Lys He Gin Val 10 1281473 1 5 10 15

Leu Gin Gin Gin Ala Asp Asp Ala Glu Glu Arg Ala Giu Arg Leu Gin 20 25 30

Arg Glu Val Glu Gly Glu Arg Arg Ala Arg Glu Gin Ala Glu Ala Glu 35 40 45

Val Ala Ser Leu Asn Arg Arg lie Gin Leu Val Glu Glu Glu Leu Asp 50 55 60

Arg Aia Gin Glu Arg Leu Ala Thr Ala Leu Gin Lys Leu Glu Glu Ala 65 70 75 80

Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met Lys Val lie Glu Asn 85 90 95

Arg Ala Leu Lys Asp Glu Glu Lys Met Glu Leu Gin Glu lie Gin Leu

100 105 110

Glu Glu Ala Lys His lie Ala Glu Glu Ala Asp Arg Lys Tyr Glu Glu 115 120 125

Val Ala Arg Lys Leu Val lie lie Glu Gly Asp Leu Glu Arg Thr Glu 130 135 · 140

Glu Arg Ala Glu Leu Ala Glu Ser Arg Cys Arg Glu Met Asp Glu Gin 145 150 155 160 lie Arg Leu Met Asp Gin Asn Leu Lys Cys Leu Ser Ala Ala Glu Glu 165 170 175

Lys Tyr Ser Gin Lys Glu Asp Lys Tyr Glu Glu Glu lie Lys lie Leu 180 185 190

Thr Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala Glu Phe Ala Glu Arg

195 200 205

Ser Val Ala Lys Leu Glu Lys Thr He Asp Asp Leu Glu Asp Thr Asn 210 215 220

Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe Gly 225 230 235 240

Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu Ser 245 250 255

Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe Gly 260 265 270 lie Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met Ser 275 280 285

Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu Gly 290 295 300

Lys Gly Ser Gly Leu Gin Gly His lie He Glu Asn Pro Gin Tyr Phe 11 1281473 305 310 315 320

Ser Asp Ala Cys Val His His lie Lys Arg Arg Asp lie Val Leu Lys 325 330 335

Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys 340 345 350

His Asn Leu Leu Pro Glu Gin Asp Lys Met Leu Val Ala Val Lys Ala 355 360 365

Leu Lys Glu Ala Ser Glu Ser Ala Arg Gin Asp Phe Gin Arg Glu Ala 370 375 380

Glu Leu Leu Thr Met Leu Gin His Gin His lie Val Arg Phe Phe Gly 385 390 395 400

Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met Arg 405 410 415

His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala Lys 420 425 430

Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu Gly 435 440 445

Gin Leu Leu Ala Val Ala Ser Gin Val Ala Ala Gly Met Val Tyr Leu 450 455 460

Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu 465 470 475 480

Val Gly Gin Gly Leu Val Val Lys lie Gly Asp Phe Gly Met Ser Arg 485 490 495

Asp lie Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met Leu 500 505 510

Pro He Arg Trp Met Pro Pro Glu Ser lie Leu Tyr Arg Lys Phe Thr 515 520 525

Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu，Ile Phe 530 535 540

Thr Tyr Gly Lys Gin Pro Trp Tyr Gin Leu Ser Asn Thr Glu Ala lie 545 550 555 560

Asp Cys lie Thr Gin Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys Pro 565 570 575

Pro Glu Val Tyr Ala He Met Arg Gly Cys Trp Gin Arg Glu Pro Ser 580 585 590

Asn Ala Thr Ala Ser Arg Met Cys Thr Pro Gly Cys Lys Pro Trp Pro 595 600 605

Arg His Leu Leu Ser Thr Trp Met Ser Trp Ala Arg Gly Pro Ala Gin 12 1281473 610 615 620

Gly Leu Gly Val Val Ser Arg Asn Thr Gly Ala Cys Pro Gin His Pro 625 630 635 640

Pro <210> 9 <211> 284 <212> PRT <213〉人屬、人種 <400> 9

Met Glu Ala lie Lys Lys Lys Met Gin Met Leu Lys Leu Asp Lys Glu 1 5 10 15

Asn Ala lie Asp Arg Ala Glu Gin Ala Glu Ala Asp Lys Lys Ala Ala 20 25 30

Glu Asp Lys Cys Lys Gin Val Glu Glu Glu Leu Thr His Leu Gin Lys 35 40 45

Lys Leu Lys Gly Thr Glu Asp Glu Leu Asp Lys Tyr Ser Glu Asp Leu 50 55 60

Lys Asp Ala Gin Glu Lys Leu Glu Leu Thr Glu Lys Lys Ala Ser Asp 65 70 75 80

Ala Glu Gly Asp Val Ala Ala Leu Asn Arg Arg lie Gin Leu Val Glu 85 90 95

Glu Glu Leu Asp Arg Ala Gin Glu Arg Leu Ala Thr Ala Leu Gin Lys 100 105 110

Leu Glu Glu Ala Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met Lys 115 120 125

Val lie Glu Asn Arg Ala Met Lys Asp Glu Glu Lys Met Glu lie Gin 130 135 140

Glu Met Gin Leu Lys Glu Ala Lys His lie Ala Glu Glu Ala Asp Arg 145 150 155 160

Lys Tyr Glu Glu Val Ala Arg Lys Leu Val lie Leu Glu Gly Glu Leu 165 170 175

Glu Arg Ala Glu Glu Arg Ala Glu Val Ser Glu Leu Lys Cys Gly Asp 180 185 190

Leu Glu Glu Glu Leu Lys Asn Val Thr Asn Asn Leu Lys Ser Leu Glu 195 200 205 13 1281473

Ala Ala Ser Glu Lys Tyr Ser Glu Lys Glu Asp Lys Tyr Glu Glu Glu 210 215 220 lie Lys Leu Leu Ser Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala Glu 225 230 235 240

Phe Ala Glu Arg Thr Val Ala Lys Leu Giu Lys Thr lie Asp Asp Leu 245 250 255

Glu Glu Lys Leu Ala Gin Ala Lys Glu Glu Asn Val Gly Leu His Gin 260 265 270

Thr Leu Asp Gin Thr Leu Asn Glu Leu Asn Cys lie 275 280

<210〉 10 <211> 417 <212〉 PRT <213〉人屬、人種 <400〉 10

Met Leu Leu Ser Val Pro Leu Leu Leu Gly Leu Leu Gly Leu Ala Val 15 10 15

Ala Glu Pro Ala Val Tyr Phe Lys Glu Gin Phe Leu Asp Gly Asp Gly 20 25 30

Trp Thr Ser Arg Trp He Glu Ser Lys His Lys Ser Asp Phe Gly Lys 35 40 45

Phe Val Leu Ser Ser Gly Lys Phe Tyr Gly Asp Glu Glu Lys Asp Lys 50 55 60

Gly Leu Gin Thr Ser Gin Asp Ala Arg Phe Tyr Ala Leu Ser Ala Ser 65 70 75 80

Phe Glu Pro Phe Ser Asn Lys Gly Gin Thr Leu Val Val Gin Phe Thr 85 90 95

Val Lys His Glu Gin Asn lie Asp Cys Gly Gly Gly Tyr Val Lys Leu 100 105 110

Phe Pro Asn Ser Leu Asp Gin Thr Asp Met His Gly Asp Ser Glu Tyr 115 120 125

Asn lie Met Phe Gly Pro Asp lie Cys Gly Pro Gly Thr Lys Lys Val 130 135 140

His Val lie Phe Asn Tyr Lys Gly Lys Asn Val Leu He Asn Lys Asp 145 150 155 160

He Arg Cys Lys Asp Asp Glu Phe Thr His Leu Tyr Thr Leu lie Val 14 1281473 165 170 175

Arg Pro Asp Asn Thr Tyr Glu Val Lys lie Asp Asn Ser Gin Val Glu 180 185 190

Ser Gly Ser Leu Glu Asp Asp Trp Asp Phe Leu Pro Pro Lys Lys lie 195 200 205

Lys Asp Pro Asp Ala Ser Lys Pro Glu Asp Trp Asp Glu Arg Ala Lys 210 215 220 lie Asp Asp Pro Thr Asp Ser Lys Pro Glu Asp Trp Asp Lys Pro Glu 225 230 235 240

His lie Pro Asp Pro Asp Ala Lys Lys Pro Glu Asp Trp Asp Glu Glu 245 250 255

Met Asp Gly Glu Trp Glu Pro Pro Val lie Gin Asn Pro Glu Tyr Lys 260 265 270

Gly Glu Trp Lys Pro Arg Gin lie Asp Asn Pro Asp Tyr Lys Gly Thr 275 280 285

Trp lie His Pro Glu lie Asp Asn Pro Glu Tyr Ser Pro Asp Pro Ser 290 295 300 lie Tyr Ala Tyr Asp Asn Phe Gly Val Leu Gly Leu Asp Leu Trp Gin 305 310 315 320

Val Lys Ser Gly Thr He Phe Asp Asn Phe Leu lie Thr Asn Asp Glu 325 330 335

Ala Tyr Ala Glu Glu Phe Gly Asn Glu Thr Trp Gly Val Thr Lys Ala 340 345 350

Ala Glu Lys Gin Met Lys Asp Lys Gin Asp Glu Glu Gin Arg Leu Lys 355 360 365

Glu Glu Glu Glu Asp Lys Lys Arg Lys Glu Glu Glu Glu Ala Glu Asp 370 375 380

Lys Glu Asp Asp Glu Asp Lys Asp Glu Asp Glu Glu Asp Glu Glu Asp 385 390 395 400

Lys Glu Glu Asp Glu Glu Glu Asp Val Pro Gly Gin Ala Lys Asp Glu 405 410 415

Leu <210> 11 <211> 278 <212〉 PRT <213〉人屬、人種 15 1281473 <400> 11

Leu Arg Cys Val Pro Arg Val Leu Gly Ser Ser Val Ala Gly Leu Arg 1 5 10 15

Ala Ala Ala Pro Ala Ser Pro Phe Arg Gin Leu Leu Gin Pro Ala Pro 20 25 30

Arg Leu Cys Thr Arg Pro Phe Gly Leu Leu Ser Val Arg Ala Gly Ser 35 40 45

Glu Arg Arg Pro Gly Leu Leu Arg Pro Arg Gly Pro Cys Ala Cys Gly 50 55 60

Cys Gly Cys Gly Ser Leu His Thr Asp Gly Asp Lys Ala Phe Val Asp 65 70 75 80

Phe Leu Ser Asp Glu lie Lys Glu Glu Arg Lys lie Gin Lys His Lys 85 90 95

Thr Leu Pro Lys Met Ser Gly Gly Trp Glu Leu Glu Leu Asn Gly Thr 100 105 110

Glu Ala Lys Leu Val Arg Lys Val Ala Gly Glu Lys lie Thr Val Thr 115 120 125

Phe Asn He Asn Asn Ser lie Pro Pro Thr Phe Asp Gly Glu Glu Glu 130 135 140

Pro Ser Gin Gly Gin Lys Val Glu Glu Gin Glu Pro Glu Leu Thr Ser 145 150 155 160

Thr Pro Asn Phe Val Val Glu Val lie Lys Asn Asp Asp Gly Lys Lys 165 170 175

Ala Leu Val Leu Asp Cys His Tyr Pro Glu Asp Glu Val Gly Gin Glu 180 185 190

Asp Glu Ala Glu Ser Asp lie Phe Ser He Arg Glu Val Ser Phe Gin 195 200 205

Ser Thr Gly Glu Ser Glu Trp Lys Asp Thr Asn Tyr Thr Leu Asn Thr 210 215 220

Asp Ser Leu Asp Trp Ala Leu Tyr Asp His Leu Met Asp Phe Leu Ala 225 230 235 240

Asp Arg Gly Val Asp Asn Thr Phe Ala Asp Glu Leu Val Glu Leu Ser 245 250 255

Thr Ala Leu Glu His Gin Glu Tyr lie Thr Phe Leu Glu Asp Leu Lys 260 265 270

Ser Phe Val Lys Ser Gin 275 16 1281473 <210> 12 <211〉 661 <212> PRT <213〉人屬、人種 <400> 12

Arg Ala Leu Arg Arg Ala Pro Ala Leu Ala Ala Val Pro Gly Gly Lys 15 10 15

Pro lie Leu Cys Pro Arg Arg Thr Thr Ala Gin Leu Gly Pro Arg Arg 20 25 30

Asn Pro Ala Trp Ser Leu Gin Ala Gly Arg Leu Phe Ser Thr Gin Thr 35 40 45

Ala Glu Asp Lys Glu Glu Pro Leu His Ser lie lie Ser Ser Thr Glu 50 55 60

Ser Val Gin Gly Ser Thr Ser Lys His Glu Phe Gin Ala Glu Thr Lys 65 70 75 80

Lys Leu Leu Asp lie Val Ala Arg Ser Leu Tyr Ser Glu Lys Glu Val 85 90 95

Phe lie Arg Glu Leu lie Ser Asn Ala Ser Asp Ala Leu Glu Lys Leu 100 105 110

Arg His Lys Leu Val Ser Asp Gly Gin Ala Leu Pro Glu Met Glu He 115 120 125

His Leu Gin Thr Asn Ala Glu Lys Gly Thr lie Thr lie Gin Asp Thr 130 135 140

Gly lie Gly Met Thr Gin Glu Glu Leu Val Ser Asn Leu Gly Thr He 145 150 155 160

Ala Arg Ser Gly Ser Lys Ala Phe Leu Asp Ala Leu Gin Asn Gin Ala 165 170 175

Glu Ala Ser Ser Lys lie lie Gly Gin Phe Gly Val Gly Phe Tyr Ser 180 185 190

Ala Phe Met Val Ala Asp Arg Val Glu Val Tyr Ser Arg Ser Ala Ala 195 200 205

Pro Gly Ser Leu Gly Tyr Gin Trp Leu Ser Asp Gly Ser Gly Val Phe 210 215 220

Glu lie Ala Glu Ala Ser Gly Val Arg Thr Gly Thr Lys lie lie lie 225 230 235 240

His Leu Lys Ser Asp Cys Lys Glu Phe Ser Ser Glu Ala Arg Val Arg 17 245 1281473 »·ν

Asp Val Val Thr Lys Tyr Ser Asn 260

Asn Gly Arg Arg Met Asn Thr Leu 275 280

Lys Asp Val Gly Glu Trp Gin His 290 295

Gin Ala His Asp Lys Pro Arg Tyr 305 310

Pro Leu Asn lie Arg Ser lie Phe 325

Met Phe Asp Val Ser Arg Glu Leu 340

Arg Lys Val Leu lie Gin Thr Lys 355 360

Leu Arg Phe lie Arg Gly Val Val 370 375

Leu Ser Arg Glu Leu Leu Gin Glu 385 390

Asp Val Leu Gin Gin Arg Leu lie 405

Lys Asp Ala Glu Lys Tyr Ala Lys 420

Met Arg Glu Gly lie Val Thr Ala 435 440 lie Ala Lys Leu Leu Arg Tyr Glu 450 455

Leu Thr Ser Leu Ser Glu Tyr Ala 465 470

Asn lie Tyr Tyr Leu Cys Ala Pro 485

Pro Tyr Tyr Glu Ala Met Lys Lys 500

Phe Glu Gin Phe Asp Glu Leu Thr 515 520

Lys Lys Lys Leu lie Ser Val Glu 530 535

Lys Glu Glu Lys Phe Glu Asp Arg 250 255

Phe Val Ser Phe Pro Leu Tyr Leu 265 270

Gin Ala lie Trp Met Met Asp Pro 285

Glu Glu Phe Tyr Arg Tyr Val Ala 300

Thr Leu His Tyr Lys Thr Asp Ala 315 320

Tyr Val Pro Asp Met Lys Pro Ser 330 335

Gly Ser Ser Val Ala Leu Tyr Ser 345 350

Ala Thr Asp He Leu Pro Lys Trp 365

Asp Ser Glu Asp lie Pro Leu Asn 380

Ser Ala Leu He Arg Lys Leu Arg 395 400

Lys Phe Phe He Asp Gin Ser Lys 410 415

Phe Phe Glu Asp Tyr Gly Leu Phe 425 430

Thr Glu Gin Glu Val Lys Glu Asp 445

Ser Ser Ala Leu Pro Ser Gly Gin 460

Ser Arg Met Arg Ala Gly Thr Arg 475 480

Asn Arg His Leu Ala Glu His Ser 490 495

Lys Asp Thr Glu Val Leu Phe Cys 505 510

Leu Leu His Leu Arg Glu Phe Asp 525

Thr Asp lie Val Val Asp His Tyr 540

Ser Pro Ala Ala Glu Cys Leu Ser

18 1281473 545 550 555 560

Glu Lys Glu Thr Glu Glu Leu Met Ala Trp Met Arg Asn Val Leu Gly 565 570 575

Ser Arg Val Thr Asn Val Lys Val Thr Leu Arg Leu Asp Thr His Pro 580 585 590

Ala Met Val Thr Val Leu Glu Met Gly Ala Ala Arg His Phe Leu Arg 595 600 605

Met Gin Gin Leu Ala Lys Thr Gin Glu Glu Arg Ala Gin Leu Leu Gin 610 615 620

Pro Thr Leu Glu lie Asn Pro Arg His Ala Leu lie Lys Lys Leu Asn 625 630 635 640

His Cys Ala Gin Ala Ser Leu Ala Trp Leu Ser Cys Trp Trp lie Arg

645 650 655

Tyr Thr Arg Thr Pro 660 <210> 13 <211〉 803 <212> PRT <213〉人屬、人種 <400> 13

Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe 15 10 15

Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu 20 25 30

Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val 35 40 45

Val Gin Arg Glu Glu Glu Ala lie Gin Leu Asp Gly Leu Asn Ala Ser 50 55 60

Gin He Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gin Ala 65 70 75 80

Glu Val Asn Arg Met Met Lys Leu lie lie Asn Ser Leu Tyr Lys Asn 85 90 95

Lys Glu lie Phe Leu Arg Glu Leu lie Ser Asn Ala Ser Asp Ala Leu 100 105 110

Asp Lys lie Arg Leu lie Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly 115 120 125 19 1281473

Asn Glu Glu Leu Thr Val Lys lie 130 135

Leu His Val Thr Asp Thr Gly Val 145 150

Lys Asn Leu Gly Thr lie Ala Lys 165

Lys Met Thr Glu Ala Gin Glu Asp 180

Gly Gin Phe Gly Val Gly Phe Tyr 195 200

Val He Val Thr Ser Lys His Asn 210 215

Ser Asp Ser Asn Glu Phe Ser Val 225 230

Leu Gly Arg Gly Thr Thr lie Thr 245

Asp Tyr Leu Glu Leu Asp Thr lie 260

Gin Phe He Asn Phe Pro lie Tyr 275 280

Val Glu Glu Pro Met Glu Glu Glu 290 295

Glu Ser Asp Asp Glu Ala Ala Val 305 310

Pro Lys Thr Lys Lys Val Glu Lys 325

Asn Asp He Lys Pro lie Trp Gin 340

Asp Glu Tyr Lys Ala Phe Tyr Lys 355 360

Pro Met Ala Tyr lie His Phe Thr 370 375

Ser lie Leu Phe Val Pro Thr Ser 385 390

Tyr Gly Ser Lys Lys Ser Asp Tyr 405

Phe lie Thr Asp Asp Phe His Asp 420

Lys Cys Asp Lys Glu Lys Asn Leu 140

Gly Met Thr Arg Glu Glu Leu Val 155 160

Ser Gly Thr Ser Glu Phe Leu Asn 170 175

Gly Gin Ser Thr Ser Glu Leu lie 185 190

Ser Ala Phe Leu Val Ala Asp Lys 205

Asn Asp Thr Gin His lie Trp Glu 220

He Ala Asp Pro Arg Gly Asn Thr 235 240

Leu Val Leu Lys Glu Glu Ala Ser 250 255

Lys Asn Leu Val Lys Lys Tyr Ser 265 270

Val Trp Ser Ser Lys Thr Glu Thr 285

Glu Ala Ala Lys Glu Glu Lys Glu 300

Glu Glu Glu Glu Glu Glu Lys Lys 315 320

Thr Val Trp Asp Trp Glu Leu Met 330 335

Arg Pro Ser Lys Glu Val Glu Glu 345 350

Ser Phe Ser Lys Glu Ser Asp Asp 365

Ala Glu Gly Glu Val Thr Phe Lys 380

Ala Pro Arg Gly Leu Phe Asp Glu 395 400 lie Lys Leu Tyr Val Arg Arg Val 410 415

Met Met Pro Lys Tyr Leu Asn Phe 425 430

20 1281473

Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg 435 440 445

Glu Thr Leu Gin Gin His Lys Leu Leu Lys Val lie Arg Lys Lys Leu 450 455 . 460

Val Arg Lys Thr Leu Asp Met lie Lys Lys lie Ala Asp Asp Lys Tyr 465 470 475 480

Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn He Lys Leu Gly Val 485 490 495 lie Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe 500 505 510

Gin Ser Ser His His Pro Thr Asp lie Thr Ser Leu Asp Gin Tyr Val 515 520 525

Glu Arg Met Lys Glu Lys Gin Asp Lys lie Tyr Phe Met Ala Gly Ser 530 535 540

Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys 545 550 555 560

Lys Gly Tyr Glu Val He Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys 565 570 575 lie Glri Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gin Asn Val Ala 580 585 590

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg 595 600 605

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp 610 615 620

Lys Ala Leu Lys Asp Lys lie Glu Lys Ala Val Val Ser Gin Arg Leu 625 630 635 640

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gin Tyr Gly Trp Ser Gly 645 650 655

Asn Met Glu Arg lie Met Lys Ala Gin Ala Tyr Gin Thr Gly Lys Asp 660 665 670

He Ser Thr Asn Tyr Tyr Ala Ser Gin Lys Lys Thr Phe Glu He Asn 675 680 685

Pro Arg His Pro Leu lie Arg Asp Met Leu Arg Arg lie Lys Glu Asp 690 695 700

Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr 705 710 715 720

Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly 725 730 735 21 1281473

Asp Arg lie Glu Arg Met Leu Arg Leu Ser Leu Asn lie Asp Pro Asp 740 745 750

Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu 755 760 765

Asp Thr Thr Glu Asp Thr Glu Gin Asp Glu Asp Glu Glu Met Asp Val 770 775 780

Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys 785 790 795 800

Asp Glu Leu

<210> 14 <211〉 724 <212〉 PRT <213〉人屬、人種 <400> 14

Met Pro Glu Glu Val His His Gly Glu Glu Glu Val Glu Thr Phe Ala 1 5 10 15

Phe Gin Ala Glu lie Ala Gin Leu Met Ser Leu lie He Asn Thr Phe 20 25 30

Tyr Ser Asn Lys Glu lie Phe Leu Arg Glu Leu lie Ser Asn Ala Ser 35 40 45

Asp Ala Leu Asp Lys lie Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys 50 55 60

Leu Asp Ser Gly Lys Glu Leu Lys lie Asp lie lie Pro Asn Pro Gin 65 70 75 80

Glu Arg Thr Leu Thr Leu Val Asp Thr Gly lie Gly Met Thr Lys Ala 85 90 95

Asp Leu lie Asn Asn Leu Gly Thr lie Ala Lys Ser Gly Thr Lys Ala 100 105 110

Phe Met Glu Ala Leu Gin Ala Gly Ala Asp lie Ser Met lie Gly Gin 115 120 125

Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val Ala Glu Lys Val Val 130 135 140

Val lie Arg Lys His Asn Asp Asp Glu Gin Tyr Ala Trp Glu Ser Ser 145 150 155 160

Ala Gly Gly Ser Phe Thr Val Arg Ala Asp His Gly Glu Pro lie Gly 22 165 1281473 δ

Met Gly Thr Lys Val lie Leu His 180

Leu Glu Glu Arg Arg Val Lys Glu 195 200 lie Gly Tyr Pro lie Thr Leu Tyr 210 215 lie Ser Asp Asp Glu Ala Glu Glu 225 230

Asp Lys Asp Asp Glu Glu Lys Pro 245

Glu Glu Asp Asp Ser Gly Lys Asp 260

Lys Glu Lys Tyr lie Asp Gin Glu 275 280

Trp Thr Arg Asn Pro Asp Asp lie 290 295

Tyr Lys Ser Leu Thr Asn Asp Trp 305 310

Phe Ser Val Glu Gly Gin Leu Glu 325

Arg Arg Ala Pro Phe Asp Leu Phe 340 lie Lys Leu Tyr Val Arg Arg Val 355 360

Leu lie Pro Glu Tyr Leu Asn Phe 370 375

Asp Leu Pro Leu Asn lie Ser Arg 385 390

Leu Lys Val lie Arg Lys Asn lie 405

Ser Glu Leu Ala Glu Asp Lys Glu 420

Phe Ser Lys Asn Leu Lys Leu Gly 435 440

Arg Arg Leu Ser Glu Leu Leu Arg 450 455

Glu Met Thr Ser Leu Ser Glu Tyr 170 175

Leu Lys Glu Asp Gin Thr Glu Tyr 185 190

Val Val Lys Lys His Ser Gin Phe 205

Leu Glu Lys Glu Arg Glu Lys Glu 220

Glu Lys Gly Glu Lys Glu Glu Glu 235 240

Lys He Glu Asp Val Gly Ser Asp 250 255

Lys Lys Lys Lys Thr Lys Lys lie 265 270

Glu Leu Asn Lys Thr Lys Pro lie 285

Thr Gin Glu Glu Tyr Gly Glu Phe 300

Glu Asp His Leu Ala Val Lys His 315 320

Phe Arg Ala Leu Leu Phe He Pro 330 335

Glu Asn Lys Lys Lys Lys Asn Asn 345 350

Phe He Met Asp Ser Cys Asp Glu 365 lie Arg Gly Val Val Asp Ser Glu 380

Glu Met Leu Gin Gin Ser Lys lie 395 400

Val Lys Lys Cys Leu Glu Leu Phe 410 415

Asn Tyr Lys Lys Phe Tyr Glu Ala 425 430 lie His Glu Asp Ser Thr Asn Arg 445

Tyr His Thr Ser Gin Ser Gly Asp 460

Val Ser Arg Met Lys Glu Thr Gin

23 1281473 465 470 475 480

Lys Ser lie Tyr Tyr lie Thr Gly Glu Ser Lys Glu Gin Val Ala Asn 485 490 495

Ser Ala Phe Val Glu Arg Val Arg Lys Arg Gly Phe Glu Val Val Tyr 500 505 510

Met Thr Glu Pro lie Asp Glu Tyr Cys Val Gin Gin Leu Lys Glu Phe 515 520 525

Asp Gly Lys Ser Leu Val Ser Val Thr Lys Glu Gly Leu Glu Leu Pro 530 535 540

Glu Asp Glu Glu Glu Lys Lys Lys Met Glu Glu Ser Lys Ala Lys Phe 545 550 555 560

Glu Asn Leu Cys Lys Leu Met Lys Glu lie Leu Asp Lys Lys Val Glu

565 570 575

Lys Val Thr lie Ser Asn Arg Leu Val Ser Ser Pro Cys Cys lie Val 580 585 590

Thr Ser Thr Tyr Gly Trp Thr Ala Asn Met Glu Arg He Met Lys Ala 595 600 605

Gin Ala Leu Arg Asp Asn Ser Thr Met Gly Tyr Met Met Ala Lys Lys 610 615 620

His Leu Glu lie Asn Pro Asp His Pro lie Val Glu Thr Leu Arg Gin 625 630 635 640

Lys Ala Glu Ala Asp Lys Asn Asp Lys Ala Val Lys Asp Leu Val Val 645 650 655

Leu Leu Phe Glu Thr Ala Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp 660 665 670

Pro Gin Thr His Ser Asn Arg lie Tyr Arg Met lie Lys Leu Gly Leu 675 680 685

Gly lie Asp Glu Asp Glu Val Ala Ala Glu Glu Pro Asn Ala Ala Val 690 695 700

Pro Asp Glu lie Pro Pro Leu Glu Gly Asp Glu Asp Ala Ser Arg Met 705 710 715 720

Glu Glu Val Asp <210> 15 <211> 732 <212> PRT <213〉人屬、人種 24 1281473 <400> 15

Met Pro Glu Glu Thr Gin Thr Gin Asp Gin Pro Met Glu Glu Glu Glu 15 10 15

Val Glu Thr Phe Ala Phe Gin Ala Glu He Ala Gin Leu Met Ser Leu 20 25 30 lie lie Asn Thr Phe Tyr Ser Asn Lys Glu He Phe Leu Arg Glu Leu 35 40 45

He Ser Asn Ser Ser Asp Ala Leu Asp Lys lie Arg Tyr Glu Thr Leu 50 55 60

Thr Asp Pro Ser Lys Leu Asp Ser Gly Lys Glu Leu His lie Asn Leu 65 70 75 80

lie Pro Asn Lys Gin Asp Arg Thr Leu Thr lie Val Asp Thr Gly lie 85 90 95

Gly Met Thr Lys Ala Asp Leu lie Asn Asn Leu Gly Thr lie Ala Lys 100 105 110

Ser Gly Thr Lys Ala Phe Met Glu Ala Leu Gin Ala Gly Ala Asp He 115 120 125

Ser Met lie Gly Gin Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val 130 135 140

Ala Glu Lys Val Thr Val lie Thr Lys His Asn Asp Asp Glu Gin Tyr 145 150 155 160

Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr Val Arg Thr Asp Thr 165 170 175

Gly Glu Pro Met Gly Arg Gly Thr Lys Val lie Leu His Leu Lys Glu 180 185 190

Asp Gin Thr Glu Tyr Leu Glu Glu Arg Arg lie Lys Glu lie Val Lys 195 200 205

Lys His Ser Gin Phe He Gly Tyr Pro He Thr Leu Phe Val Glu Lys 210 215 220

Glu Arg Asp Lys Glu Val Ser Asp Asp Glu Ala Glu Glu Lys Glu Asp 225 230 235 240

Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu Ser Glu Asp Lys Pro 245 250 255

Glu lie Glu Asp Val Gly Ser Asp Glu Glu Glu Glu Lys Lys Asp Gly 260 265 270

Asp Lys Lys Lys Lys Lys Lys lie Lys Glu Lys Tyr lie Asp Gin Glu 275 280 285 25 1281473

Glu Leu Asn Lys Thr Lys Pro lie Trp Thr Arg Asn Pro Asp Asp He 290 295 300

Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu Thr Asn Asp Trp 305 310 315 320

Glu Asp His Leu Ala Val Lys His Phe Ser Val Glu Gly Gin Leu Glu 325 330 335

Phe Arg Ala Leu Leu Phe Val Pro Arg Arg Ala Pro Phe Asp Leu Phe 340 345 350

Glu Asn Arg Lys Lys Lys Asn Asn He Lys Leu Tyr Val Arg Arg Val 355 360 365

Phe lie Met Asp Asn Cys Glu Glu Leu lie Pro Glu Tyr Leu Asn Phe 370 375 380

lie Arg Gly Val Val Asp Ser Glu Asp Leu Pro Leu Asn He Ser Arg 385 390 395 400

Glu Met Leu Gin Gin Ser Lys lie Leu Lys Val lie Arg Lys Asn Leu 405 410 415

Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Leu Ala Glu Asp Lys Glu 420 425 430

Asn Tyr Lys Lys Phe Tyr Glu Gin Phe Ser Lys Asn lie Lys Leu Gly 435 440 445 lie His Glu Asp Ser Gin Asn Arg Lys Lys Leu Ser Glu Leu Leu Arg 450 455 460

Tyr Tyr Thr Ser Ala Ser Gly Asp Glu Met Val Ser Leu Lys Asp Tyr 465 470 475 480

Cys Thr Arg Met Lys Glu Asn Gin Lys His lie Tyr Tyr lie Thr Gly 485 490 495

Glu Thr Lys Asp Gin Val Ala Asn Ser Ala Phe Val Glu Arg Leu Arg 500 505 510

Lys His Gly Leu Glu Val lie Tyr Met He Glu Pro lie Asp Glu Tyr 515 520 525

Cys Val Gin Gin Leu Lys Glu Phe Glu Gly Lys Thr Leu Val Ser Val 530 535 540

Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu Glu Lys Lys Lys 545 550 555 560

Gin Glu Glu Lys Lys Thr Lys Phe Glu Asn Leu Cys Lys lie Met Lys 565 570 575

Asp lie Leu Glu Lys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu 580 585 590 26 J281473

Val Thr Ser Pro Cys Cys lie Val Thr Ser Thr Tyr Gly Trp Thr Ala 595 600 605

Asn Met Glu Arg lie Met Lys Ala Gin Ala Leu Arg Asp Asn Ser Thr 610 615 620

Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu He Asn Pro Asp His 625 630 635 640

Ser lie lie Glu Thr Leu Arg Gin Lys Ala Glu Ala Asp Lys Asn Asp 645 650 655

Lys Ser Val Lys Asp Leu Val He Leu Leu Tyr Glu Thr Ala Leu Leu 660 665 670

Ser Ser Gly Phe Ser Leu Glu Asp Pro Gin Thr His Ala Asn Arg lie 675 680 685

Tyr Arg Met lie Lys Leu Gly Leu Gly lie Asp Glu Asp Asp Pro Thr 690 695 700

Ala Asp Asp Thr Ser Ala Ala Val Thr Glu Glu Met Pro Pro Leu Glu 705 710 715 720

Gly Asp Asp Asp Thr Ser Arg Met Glu Glu Val Asp 725 730 <210>. 16 <211〉 573 <212> PRT <213〉人屬、人種

<400〉 16

Met Leu Arg Leu Pro Thr Val Phe Arg Gin Met Arg Pro Val Ser Arg 1 5 10 . 15

Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20 25 30

Gly Ala Asp Ala Arg Ala Leu Met Leu Gin Gly Val Asp Leu Leu Ala 35 40 45

Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr Val He lie 50 55 60

Glu Gin Ser Trp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val 65 70 75 80

Ala Lys Ser lie Asp Leu Lys Asp Lys Tyr Lys Asn lie Gly Ala Lys 85 90 95

Leu Val Gin Asp Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly 27 100 1281473

Thr Thr Thr Ala Thr Val Leu Ala 115 120

Glu Lys lie Ser Lys Gly Ala Asn 130 135

Met Leu Ala Val Asp Ala Val lie 145 150

Pro Val Thr Thr Pro Glu Glu lie 165

Asn Gly Asp Lys Glu He Gly Asn 180

Val Gly Arg Lys Gly Val lie Thr 195 200

Asp Glu Leu Glu lie lie Glu Gly 210 215

Ser Pro Tyr Phe lie Asn Thr Ser 225 230

Asp Ala Tyr Val Leu Leu Ser Glu 245 lie Val Pro Ala Leu Glu lie Ala 260 lie lie Ala Glu Asp Val Asp Gly 275 280

Asn Arg Leu Lys Val Gly Leu Gin 290 295

Phe Gly Asp Asn Arg Lys Asn Gin 305 310

Gly Gly Ala Val Phe Gly Glu Glu 325

Val Gin Pro His Asp Leu Gly Lys 340

Asp Asp Ala Met Leu Leu Lys Gly 355 360

Lys Arg lie Gin Glu lie lie Glu 370 375

Tyr Glu Lys Glu Lys Leu Asn Glu 385 390

Val Ala Val Leu Lys Val Gly Gly 105 110

Arg Ser lie Ala Lys Glu Gly Phe 125

Pro Val Glu lie Arg Arg Gly Val 140

Ala Glu Leu Lys Lys Gin Ser Lys 155 160

Ala Gin Val Ala Thr lie Ser Ala 170 175 lie He Ser Asp Ala Met Lys Lys 185 190

Val Lys Asp Gly Lys Thr Leu Asn 205

Met Lys Phe Asp Arg Gly Tyr lie 220

Lys Gly Gin Lys Cys Glu Phe Gin 235 240

Lys Lys lie Ser Ser lie Gin Ser 250 255

Asn Ala His Arg Lys Pro Leu Val 265 270

Glu Ala Leu Ser Thr Leu Val Leu 285

Val Val Ala Val Lys Ala Pro Gly 300

Leu Lys Asp Met Ala He Ala Thr 315 320

Gly Leu Thr Leu Asn Leu Glu Asp 330 335

Val Gly Glu Val He Val Thr Lys 345 350

Lys Gly Asp Lys Ala Gin lie Glu 365

Gin Leu Asp Val Thr Thr Ser Glu 380

Arg Leu Ala Lys Leu Ser Asp Gly 395 400

Thr Ser Asp Val Glu Val Asn Glu

28 1281473 405 410 415

Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val 420 425 430

Glu Glu Gly lie Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys lie 435 440 445

Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp Gin Lys He Gly 450 455 460 lie Glu lie He Lys Arg Thr Leu Lys He Pro Ala Met Thr He Ala 465 470 475 480

Lys Asn Ala Gly Val Glu Gly Ser Leu lie Val Glu Lys lie Met Gin 485 490 495

Ser Ser Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn 500 505 510

Met Val Glu Lys Gly lie lie Asp Pro Thr Lys Val Val Arg Thr Ala 515 520 525

Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala Glu Val 530 535 540

Val Val Thr Glu lie Pro Lys Glu Glu Lys Asp Pro Gly Met Gly Ala 545 550 555 560

Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly Met Phe 565 570 <210〉 17 <211> 215 <212〉 PRT <213〉人屬、人種 <400> 17 _

Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Arg Lys Met Ser Ser Tyr 15 10 15

Ala Phe Phe Val Gin Thr Cys Arg Glu Glu His Lys Lys Lys His Pro 20 25 30

Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg 35 40 45

Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala 50 55 60

Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr lie Pro 65 70 75 80 29 1281473

Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys 85 90 95

Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys 100 105 110 lie Lys Gly Glu His Pro Gly Leu Ser He Gly Asp Val Ala Lys Lys 115 120 125

Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gin Pro Tyr 130 135 140

Glu Lys Lys Ala Glu Lys Leu Lys Glu Lys Tyr Glu Lys Asp lie Ala 145 150 155 160

Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val 165 170 175

Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Gly Glu Glu 180 185 190

Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu 195 200 205

Glu Glu Asp Asp Asp Asp Glu 210 215 <210> 18 <211> 1087 <212〉 PRT <213〉人屬、人種

<400> 18

Met Met Thr Ser Val Gly Thr Asn Arg Ala Arg Gly Asn Trp Glu Gin 15 10 15

Pro Gin Asn Gin Asn Gin Thr Gin His Lys Gin Arg Pro Gin Ala Thr 20 25 30

Ala Glu Gin lie Arg Leu Ala Gin Met lie Ser Asp His Asn Asp Ala 35 40 45

Asp Phe Glu Glu Lys Val Lys Gin Leu lie Asp lie Thr Gly Lys Asn 50 55 60

Gin Asp Glu Cys Val lie Ala Leu His Asp Cys Asn Gly Asp Val Asn 65 70 75 80

Arg Ala lie Asn Val Leu Leu Glu Gly Asn Pro Asp Thr His Ser Trp 85 90 95

Glu Met Val Gly Lys Lys Lys Gly Val Ser Gly Gin Lys Asp Gly Gly 30 ..、s 1281473 100 105 110

Gin Thr Glu Ser Asn Glu Glu Gly Lys Glu Asn Arg Asp Arg Asp Arg 115 120 125

Asp Tyr Ser Arg Arg Arg Gly Gly Pro Pro Arg Arg Gly Arg Gly Ala 130 135 140

Ser Arg Gly Arg Glu Phe Arg Gly Gin Glu Asn Gly Leu Asp Gly Thr 145 150 155 160

Lys Ser Gly Gly Pro Ser Gly Arg Gly Thr Glu Arg Gly Arg Arg Gly 165 170 175

Arg Gly Arg Gly Arg Gly Gly Ser Gly Arg Arg Gly Gly Arg Phe Ser 180 185 190

Ala Gin Gly Met Gly Thr Phe Asn Pro Ala Asp Tyr Ala Glu Pro Ala 195 200 205

Asn Thr Asp Asp Asn Tyr Gly Asn Ser Ser Gly Asn Thr Trp Asn Asn 210 215 220

Thr Gly His Phe Glu Pro Asp Asp Gly Thr Ser Ala Trp Arg Thr Ala 225 230 235 240

Thr Glu Glu Trp Gly Thr Glu Asp Trp Asn Glu Asp Leu Ser Glu Thr 245 250 255

Lys lie Phe Thr Ala Ser Asn Val Ser Ser Val Pro Leu Pro Ala Glu 260 265 270

Asn Val Thr lie Thr Ala Gly Gin Arg lie Asp Leu Ala Val Leu Leu 275 280 285

Gly Lys Thr Pro Ser Thr Met Glu Asn Asp Ser Ser Asn Leu Asp Pro 290 295 300

Ser Gin Ala Pro Ser Leu Ala Gin Pro Leu Val Phe Ser Asn Ser Lys 305 310 315 320

Gin Thr Ala He Ser Gin Pro Ala Ser Gly Asn Thr Phe Ser His His 325 330 335

Ser Met Val Ser Met Leu Gly Lys Gly Phe Gly Asp Val Gly Glu Ala 340 345 350

Lys Gly Gly Ser Thr Thr Gly Ser Gin Phe Leu Glu Gin Phe Lys Thr 355 360 365

Ala Gin Ala Leu Ala Gin Leu Ala Ala Gin His Ser Gin Ser Giy Ser 370 375 380

Thr Thr Thr Ser Ser Trp Asp Met Gly Ser Thr Thr Gin Ser Pro Ser 385 390 395 400

Leu Val Gin Tyr Asp Leu Lys Asn Pro Ser Asp Ser Ala Val His Ser 31 405 1281473

Pro Phe Thr Lys Arg Gin Ala Phe 420

Val Phe Leu Gin Glu Lys Ser Pro 435 440

Pro Pro Pro Pro Ser Ser Pro Leu 450 455

Gin Met Ser Pro Gly Ser Ser Asp 465 470

Ala His Gin Lys Leu Lys Gin Gin 485

Lys lie Pro Ala Leu Ala Val Glu 500

Gly Leu Asn Leu Gin Phe Gly Ala 515 520

Leu Ser Asp Tyr Glu Ser Thr Pro 530 535

Ala Pro Ser Ser Leu Tyr Thr Ser 545 550

He Ser Ser Asn Gin Ser Gin Glu 565

Gin Ser Thr Thr Tyr Thr Ser Gin 580

Glu Gin Arg Ser Thr Gin Thr Arg 595 600

Ser Pro Gin Lys Asp Leu Thr Gin 610 615

Gin Ala Thr Gin Leu Gin Thr Thr 625 630

Ser Ala Val Lys Ser Asp Ser Pro 645

Asn Glu Thr Val Ser Ala Ala Ser 660

Ser Ser Ser Leu Gly Gly Leu Ser 675 680

Thr Thr Thr Gin His Ser Ser Thr 690 695

Ser Ser Ser Thr Ser Ser Gly Arg 410 415

Thr Pro Ser Ser Thr Met Met Glu 425 430

Ala Val Ala Thr Ser Thr Ala Ala 445

Pro Ser Lys Ser Thr Ser Ala Pro 460

Asn Gin Ser Ser Ser Pro Gin Pro 475 480

Lys Lys Lys Ala Ser Leu Thr Ser 490 495

Met Pro Gly Ser Ala Asp lie Ser 505 510

Leu Gin Phe Gly Ser Glu Pro Val 525

Thr Thr Ser Ala Ser Ser Ser Gin 540

Thr Ala Ser Glu Ser Ser Ser Thr 555 560

Ser Gly Tyr Gin Ser Gly Pro lie 570 575

Asn Asn Ala Gin Gly Pro Leu Tyr 585 590

Arg Tyr Pro Ser Ser lie Ser Ser 605

Ala Lys Asn Gly Phe Ser Ser Val 620

Gin Ser Val Glu Gly Ala Thr Gly 635 640

Ser Thr Ser Ser lie Pro Pro Leu 650 655

Leu Leu Thr Thr Thr Asn Gin His 665 670

His Ser Glu Glu lie Pro Asn Thr 685

Leu Ser Thr Gin Gin Asn Thr Leu 700

Thr Ser Thr Ser Thr Leu Leu His

32 1281473 、 705 710 715 720

Thr Ser Val Glu Ser Glu Ala Asn Leu His Ser Ser Ser Ser Thr Phe 725 730 735

Ser Thr Thr Ser Ser Thr Val Ser Ala Pro Pro Pro Val Val Ser Val 740 745 750

Ser Ser Ser Leu Asn Ser Gly Ser Ser Leu Gly Leu Ser Leu Gly Ser 755 760 765

Asn Ser Thr Val Thr Ala Ser Thr Arg Ser Ser Val Ala Thr Thr Ser 770 775 780

Gly Lys Ala Pro Pro Asn Leu Pro Pro Gly Val Pro Pro Leu Leu Pro 785 790 795 800

Asn Pro Tyr He Met Ala Pro Gly Leu Leu His Ala Tyr Pro Pro Gin 805 810 815

Val Tyr Gly Tyr Asp Asp Leu Gin Met Leu Gin Thr Arg Phe Pro Leu 820 825 830

Asp Tyr Tyr Ser lie Pro Phe Pro Thr Pro Thr Thr Pro Leu Thr Gly 835 840 845

Arg Asp Gly Ser Leu Ala Ser Asn Pro Tyr Ser Gly Asp Leu Thr Lys 850 855 860

Phe Gly Arg Gly Asp Ala Ser Ser Pro Ala Pro Ala Thr Thr Leu Ala 865 870 875 880

Gin Pro Gin Gin Asn Gin Thr Gin Thr His His Thr Thr Gin Gin Thr 885 890 895

Phe Leu Asn Pro Ala Leu Pro Pro Gly Tyr Ser Tyr Thr Ser Leu Pro 900 905 910

Tyr Tyr Thr Gly Val Pro Gly Leu Pro Ser Thr Phe Gin Tyr Gly Pro 915 920 925

Ala Val Phe Pro Val Ala Pro Thr Ser Ser Lys Gin His Gly Val Asn 930 935 940

Val Ser Val Asn Ala Ser Ala Thr Pro Phe Gin Gin Pro Ser Gly Tyr 945 950 955 960

Gly Ser His Gly Tyr Asn Thr Gly Val Ser Val Thr Ser Ser Asn Thr 965 970 975

Gly Val Pro Asp lie Ser Gly Ser Val Tyr Ser Lys Thr Gin Gin Ser 980 985 990

Phe Glu Lys Gin Gly Phe His Ser Gly Thr Pro Ala Ala Ser Phe Asn 995 1000 1005

Leu Pro Ser Ala Leu Gly Ser Gly Gly Pro lie Asn Pro Ala Thr 33 1281473 1010 1015 1020

Ala Ala Ala Tyr Pro Pro Ala Pro Phe Met His lie Leu Thr Pro 1025 1030 1035

His Gin Gin Pro His Ser Gin lie Leu His His His Leu Gin Gin 1040 1045 1050

Asp Gly Gin Thr Gly Ser Gly Gin Arg Ser Gin Thr Ser Ser lie 1055 1060 1065

Pro Gin Lys Pro Gin Thr Asn Lys Ser Ala Tyr Asn Ser Tyr Ser 1070 1075 1080

Trp Gly Ala Asn 1085

<210> 19 <211〉 806 <212〉 PRT <213〉人屬、人種 <400> 19

Met Ala Ser Gly Ala Asp Ser Lys Gly Asp Asp Leu Ser Thr Ala He 1 5 . 10 15

Leu Lys Gin Lys Asn Arg Pro Asn Arg Leu lie Val Asp Glu Ala lie 20 25 30

Asn Glu Asp Asn Ser Val Val Ser Leu Ser Gin Pro Lys Met Asp Glu 35 40 45

Leu Gin Leu Phe Arg Gly Asp Thr Val Leu Leu Lys Gly Lys Lys Arg 50 55 60

Arg Glu Ala Val Cys He Val Leu Ser Asp Asp Thr Cys Ser Asp Glu 65 70 75 ' 80

Lys lie Arg Met Asn Arg Val Val Arg Asn Asn Leu Arg Val Arg Leu 85 90 95

Gly Asp Val lie Ser He Gin Pro Cys Pro Asp Val Lys Tyr Gly Lys 100 105 110

Arg lie His Val Leu Pro lie Asp Asp Thr Val Glu Gly lie Thr Gly 115 120 125

Asn Leu Phe Glu Val Tyr Leu Lys Pro Tyr Phe Leu Glu Ala Tyr Arg 130 135 140

Pro lie Arg Lys Gly Asp lie Phe Leu Val Arg Gly Gly Met Arg Ala 145 150 155 160 34 1281473

Val Glu Phe Lys Val Val Glu Thr Asp Pro Ser Pro Tyr Cys lie Val 165 170 175

Ala Pro Asp Thr Val lie His Cys Glu Gly Glu Pro lie Lys Arg Glu 180 185 190

Asp Glu Glu Glu Ser Leu Asn Glu Val Gly Tyr Asp Asp lie Gly Gly 195 200 205

Cys Arg Lys Gin Leu Ala Gin lie Lys Glu Met Val Glu Leu Pro Leu 210 215 220

Arg His Pro Ala Leu Phe Lys Ala He Gly Val Lys Pro Pro Arg Gly 225 230 235 240 lie Leu Leu Tyr Gly Pro Pro Gly Thr Gly Lys Thr Leu He Ala Arg 245 250 255

Ala Val Ala Asn Glu Thr Gly Ala Phe Phe Phe Leu lie Asn Gly Pro 260 265 270

Glu lie Met Ser Lys Leu Ala Gly Glu Ser Glu Ser Asn Leu Arg Lys 275 280 285

Ala Phe Glu Glu Ala Glu Lys Asn Ala Pro Ala lie He Phe lie Asp 290 295 300

Glu Leu Asp Ala lie Ala Pro Lys Arg Glu Lys Thr His Gly Glu Val 305 310 315 320

Glu Arg Arg lie Val Ser Gin Leu Leu Thr Leu Met Asp Gly Leu Lys 325 330 335

Gin Arg Ala His Val lie Val Met Ala Ala Thr Asn Arg Pro Asn Ser 340 345 350

He Asp Pro Ala Leu Arg Arg Phe Gly Arg Phe Asp Arg Glu Val Asp 355 360 365 lie Gly lie Pro Asp Ala Thr Gly Arg Leu Glu lie Leu Gin He His 370 375 380

Thr Lys Asa Met Lys Leu Ala Asp Asp Val Asp Leu Glu Gin Val Ala 385 390 395 400

Asn Glu Thr His Gly His Val Gly Ala Asp Leu Ala Ala Leu Cys Ser 405 410 415

Glu Ala Ala Leu Gin Ala lie Arg Lys Lys Met Asp Leu lie Asp Leu 420 425 430

Glu Asp Glu Thr lie Asp Ala Glu Val Met Asn Ser Leu Ala Val Thr 435 440 445

Met Asp Asp Phe Arg Trp Ala Leu Ser Gin Ser Asn Pro Ser Ala Leu 450 455 460 35 1281473

Arg Glu Thr Val Val Giu Val Pro Gin Val Thr Trp Glu Asp lie Gly 465 470 475 480

Gly Leu Glu Asp Val Lys Arg Glu Leu Gin Glu Leu Val Gin Tyr Pro 485 490 495

Val Glu His Pro Asp Lys Phe Leu Lys Phe Gly Met Thr Pro Ser Lys 500 505 510

Gly Val Leu Phe Tyr Gly Pro Pro Gly Cys Gly Lys Thr Leu Leu Ala 515 520 525

Lys Ala He Ala Asn Glu Cys Gin Ala Asn Phe He Ser lie Lys Gly 530 535 540

Pro Glu Leu Leu Thr Met Trp Phe Gly Glu Ser Glu Ala Asn Val Arg 545 550 555 560

Glu lie Phe Asp Lys Ala Arg Gin Ala Ala Pro Cys Val Leu Phe Phe 565 570 575

Asp Glu Leu Asp Ser lie Ala Lys Ala Arg Gly Gly Asn He Gly Asp 580 585 590

Gly Gly Gly Ala Ala Asp Arg Val lie Asn Gin lie Leu Thr Glu Met 595 600 605

Asp Gly Met Ser Thr Lys Lys Asn Val Phe lie He Gly Ala Thr Asn 610 615 620

Arg Pro Asp lie lie Asp Pro Ala He Leu Arg Pro Gly Arg Leu Asp 625 630 635 640

Gin Leu He Tyr lie Pro Leu Pro Asp Glu Lys Ser Arg Val Ala He 645 650 655

Leu Lys Ala Asn Leu Arg Lys Ser Pro Val Ala Lys Asp Val Asp Leu 660 665 670

Glu Phe Leu Ala Lys Met Thr Asn Gly Phe Ser Gly Ala Asp Leu Thr 675 680 685

Glu lie Cys Gin Arg Ala Cys Lys Leu Ala lie Arg Glu Ser lie Glu 690 695 700

Ser Glu lie Arg Arg Glu Arg Glu Arg Gin Thr Asn Pro Ser Ala Met 705 710 715 720

Glu Val Glu Glu Asp Asp Pro Val Pro Glu lie Arg Arg Asp His Phe 725 730 735

Glu Glu Ala Met Arg Phe Ala Arg Arg Ser Val Ser Asp Asn Asp He 740 745 750

Arg Lys Tyr Glu Met Phe Ala Gin Thr Leu Gin Gin Ser Arg Gly Phe 755 760 765 36 1281473

Gly Ser Phe Arg Phe Pro Ser Gly Asn Gin Gly Gly Ala Gly Pro Ser 770 775 780

Gin Gly Ser Gly Gly Gly Thr Gly Gly Ser Val Tyr Thr Glu Asp Asn 785 790 795 800

Asp Asp Asp Leu Tyr Gly 805 <210> 20 <211〉 260 <212> PRT <213〉人屬、人種

<400〉 20

Met Val Lys Val Lys Val Glu Val Asn Gly Phe Gly His Thr Gly Arg 15 10 15

Leu Val Thr Arg Ala Ala Phe Asn Ser Gly Lys Val Asp lie Val Thr 20 25 30 lie Asn Asp Pro Phe He Asp Leu Asn Tyr Pro Phe lie Asp Leu Asn 35 40 45

Tyr Met lie Tyr Met Phe Gin Tyr Asp Ser Met Ala Asn Ser Met Ala 50 55 60

Pro Ser Arg Leu Arg Met Gly Ser Leu Ser Ser Arg Glu lie Pro Ser 65 70 75 80

Pro Ser Ser Arg Ser Glu lie Pro Pro Lys Ser Asn Gly Gly Glu Ala

85 90 95

Lys Arg lie lie He Ser Ala Pro Ser Ala Asp Ala Pro Met Phe Met 100 105 110

Met Gly lie Asn Arg Glu Lys Tyr Asp Asn Ser Leu Glu lie He Ser 115 120 125

Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys Val lie 130 135 140

His Asp Asn Phe Gly lie Met Glu Gly Leu Met Thr Thr Val His Ala 145 150 155 160 lie Ala Ala Thr Gin Lys Thr Val Asp Ser Pro Ser Gly Lys Leu Trp 165 170 175

Cys Asp Gly Cys Arg Ala Leu Gin Asn lie lie Pro Ala Ser Thr Gly 180 185 190

Ser Ser Leu Ala Lys Pro Ser Val Val Pro Thr Thr Asn Val Ser Val 37 1281473 195 200 205

Val Asp Leu Thr Cys His Leu Glu Lys Pro Ala Lys Tyr Asp Asp He 210 215 220

Lys Lys Trp Cys Ser Arg His Gin Lys Ala Pro Ser Arg Ala Ser Trp 225 230 235 240

Ala Thr Leu Ser Thr Cys Asn Arg Val Val Asp Leu Met Ala His Met 245 250 255

Ala Ser Lys Glu 260 <210〉 21 <211〉 279 <212> PRT · <213〉人屬、人種 <400> 21

Met Asn Lys Val Glu Leu Glu Ser Arg Leu Glu Gly Leu Thr Asp Glu 1 5 10 15 - lie Asn Phe Leu Arg Gin Leu Tyr Glu Glu Glu Leu Arg Glu Leu Gin 20 25 30

Ser Gin lie Ser Asp Thr Ser Val Val Leu Ser Met Asp Asn Ser Arg 35 40 45

Ser Leu Asp Met Asp Ser lie He Ala Glu Val Lys Ala Gin Tyr Glu 50 55 60

Asp lie Ala Asn Arg Ser Arg Ala Glu Ala Glu Ser Met Tyr Gin lie 65 70 75 80 ♦

Lys Tyr Glu Glu Leu Gin Ser Leu Ala Gly Lys His Gly Asp Asp Leu 85 90 、95

Arg Arg Thr Lys Thr Glu lie Ser Glu Met Asn Arg Asn lie Ser Arg 100 105 110

Leu Gin Ala Glu lie Glu Gly Leu Lys Gly Gin Arg Ala Ser Leu Glu 115 120 125

Ala Ala lie Ala Asp Ala Glu Gin Arg Gly Glu Leu Ala lie Lys Asp 130 135 140

Ala Asn Ala Lys Leu Ser Glu Leu Glu Ala Ala Leu Gin Arg Ala Lys 145 150 155 160

Gin Asp Met Ala Arg Gin Leu Arg Glu Tyr Gin Glu Leu Met Asn Val 165 170 175 38 1281473

Lys Leu Ala Met Asp lie Glu lie Ala Thr Tyr Arg Lys Leu Leu Glu 180 185 190

Gly Glu Glu Ser Arg Leu Glu Ser Gly Met Gin Asa Met Ser lie His 195 200 205

Thr Lys Thr Thr Gly Gly Tyr Ala Gly Gly Leu Ser Ser Ala Tyr Gly 210 215 220

Gly Leu Ala Ser Pro Gly Leu Ser Tyr Ser Leu Gly Ser Ser Phe Gly 225 230 235 240

Ser Gly Ala Gly Ser Ser Ser Phe Ser Arg Thr Ser Ser Ser Arg Ala 245 250 255

Val Val Val Lys Lys lie Glu Thr Arg Asp Gly Lys Leu Val Ser Glu 260 265 270

Ser Ser Asp Val Leu Pro Lys 275 <210> 22 <211〉 577 <212> PRT <213〉人屬、人種 <400> 22

Met Asn Lys Leu Tyr lie Gly Asn Leu Asn Glu Ser Val Thr Pro Ala 15 10 15

Asp Leu Glu Lys Val Phe Ala Glu His Lys lie Ser Tyr Ser Gly Gin 20 25 30

Phe Leu Val Lys Ser Gly Tyr Ala Phe Val Asp Cys Pro Asp Glu His 35 40 45

Trp Ala Met Lys Ala lie Glu Thr Phe Ser Gly Lys Val Glu Leu Gin 50 55 60

Gly Lys Arg Leu Glu lie Glu His Ser Val Pro Lys Lys Gin Arg Ser 65 70 75 80

Arg Lys lie Gin He Arg Asn lie Pro Pro Gin Leu Arg Trp Glu Val 85 90 95

Leu Asp Ser Leu Leu Ala Gin Tyr Gly Thr Val Glu Asn Cys Glu Gin 100 105 110

Val Asn Thr Glu Ser Glu Thr Ala Val Val Asn Val Thr Tyr Ser Asn 115 120 125

Arg Glu Gin Thr Arg Gin Ala He Met Lys Leu Asn Gly His Gin Leu 39 1281473 130 135 140

Glu Asn His Ala Leu Lys Val Ser Tyr lie Pro Asp Glu Gin lie Ala 145 150 155 160

Gin Gly Pro Glu Asn Gly Arg Arg Gly Gly Phe Gly Ser Arg Gly Gin 165 170 175

Pro Arg Gin Gly Ser Pro Val Ala Ala Gly Ala Pro Ala Lys Gin Gin 180 185 190

Gin Val Asp He Pro Leu Arg Leu Leu Val Pro Thr Gin Tyr Val Gly 195 200 205

Ala lie lie Gly Lys Glu Gly Ala Thr He Arg Asn lie Thr Lys Gin 210 215 220

Thr Gin Ser Lys lie Asp Val His Arg Lys Glu Asn Ala Gly Ala Ala

225 230 235 240

Glu Lys Ala lie Ser Val His Ser Thr Pro Glu Gly Cys Ser Ser Ala 245 250 255

Cys Lys Met He Leu Glu lie Met His Lys Glu Ala Lys Asp Thr Lys 260 265 270

Thr Ala Asp Glu Val Pro Leu Lys lie Leu Ala His Asn Asn Phe Val 275 280 285

Gly Arg Leu lie Gly Lys Glu Gly Arg Asn Leu Lys Lys Val Glu Gin 290 295 300

Asp Thr Glu Thr Lys He Thr He Ser Ser Leu Gin Asp Leu Thr Leu 305 310 315 320

Tyr Asn Pro Glu Arg Thr lie Thr Val Lys Gly Ala He Glu Asa Cys

325 330 335

Cys Arg Ala Glu Gin Glu lie Met Lys Lys Val Arg Glu Ala Tyr Glu 340 345 350

Asn Asp Val Ala Ala Met Ser Leu Gin Ser His Leu lie Pro Gly Leu 355 360 365

Asn Leu Ala Ala Val Gly Leu Phe Pro Ala Ser Ser Ser Ala Val Pro 370 375 380

Pro Pro Pro Ser Ser Val Thr Gly Ala Ala Pro Tyr Ser Ser Phe Met 385 390 395 400

Gin Ala Pro Glu Gin Glu Met Val Gin Val Phe lie Pro Ala Gin Ala 405 410 415

Val Gly Ala lie He Gly Lys Lys Gly Gin His He Lys Gin Leu Ser 420 425 430

Arg Phe Ala Ser Ala Ser lie Lys He Ala Pro Pro Glu Thr Pro Asp 40 1281473 435 440 445

Ser Lys Val Arg Met Val lie lie Thr Gly Pro Pro Glu Ala Gin Phe 450 455 460

Lys Ala Gin Gly Arg lie Tyr Gly Lys Leu Lys Glu Glu Asn Phe Phe 465 470 475 480

Gly Pro Lys Glu Glu Val Lys Leu Glu Thr His lie Arg Val Pro Ala 485 490 495

Ser Ala Ala Gly Arg Val lie Gly Lys Gly Gly Lys Thr Val Asn Glu 500 505 510

Leu Gin Asn Leu Thr Ala Ala Glu Val Val Val Pro Arg Asp Gin Thr 515 520 525

Pro Asp Glu Asn Asp Gin Val lie Val Lys lie lie Gly His Phe Tyr 530 535 540

Ala Ser Gin Met Ala Gin Arg Lys lie Arg Asp He Leu Ala Gin Val 545 550 555 560

Lys Gin Gin His Gin Lys Gly Gin Ser Asn Gin Ala Gin Ala Arg Arg 565 570 575

Lys <210> 23 <211> 329 <212> PRT <213〉人屬、人種 <400> 23

Met Ala Thr Gly Gin Lys Leu Met Arg Ala Val Arg Val Phe Glu Phe 15 10 15

Gly Gly Pro Glu Val Leu Lys Leu Arg Ser Asp He Ala Val Pro lie 20 25 30

Pro Lys Asp His Gin Val Leu lie Lys Val His Ala Cys Gly Val Asn 35 40 45

Pro Val Glu Thr Tyr lie Arg Ser Gly Thr Tyr Ser Arg Lys Pro Leu 50 55 60

Leu Pro Tyr Thr Pro Gly Ser Asp Val Ala Gly Val lie Glu Ala Val 65 70 75 80

Gly Asp Asn Ala Ser Ala Phe Lys Lys Gly Asp Arg Val Phe Thr Ser 85 90 95 41 .·Ί 1281473

Ser Thr lie Ser Gly Gly Tyr Ala Glu Tyr Ala Leu Ala Ala Asp His 100 105 110

Thr Val Tyr Lys Leu Pro Glu Lys Leu Asp Phe Lys Gin Gly Ala Ala 115 120 125 lie Gly lie Pro Tyr Phe Thr Ala Tyr Arg Ala Leu lie His Ser Ala 130 135 140

Cys Val Lys Ala Gly Glu Ser Val Leu Val His Gly Ala Ser Gly Gly 145 150 155 160

Val Gly Leu Ala Ala Cys Gin He Ala Arg Ala Tyr Gly Leu Lys lie 165 170 175

Leu Gly Thr Ala Gly Thr Glu Glu Gly Gin Lys lie Val Leu Gin Asn 180 185 190‘

Gly Ala His Glu Val Phe Asn His Arg Glu Val Asn Tyr He Asp Lys 195 200 205 lie Lys Lys Tyr Val Gly Glu Lys Gly lie Asp lie lie He Glu Met 210 215 220

Leu Ala Asn Val Asn Leu Ser Lys Asp Leu Ser Leu Leu Ser His Gly 225 230 235 240

Gly Arg Val He Val Val Gly Ser Arg Gly Thr lie Glu lie Asn Pro 245 . 250 255

Arg Asp Thr Met Ala Lys Glu Ser Ser lie lie Gly Val Thr Leu Phe 260 265 270

Ser Ser Thr Lys Glu Glu Phe Gin Gin Tyr Ala Ala Ala Leu Gin Ala 275 280 285

Gly Met Glu lie Gly Trp Leu Lys Pro Val lie Gly Ser Gin Tyr Pro 290 295 300

Leu Glu Lys Val Ala Glu Ala His Glu Asn lie lie His Gly Ser Gly 305 310 315 320

Ala Thr Gly Lys Met lie Leu Leu Leu 325 . <210> 24 <211> 125 <212〉 PRT <213〉人屬、人種 <400> 24

Met Gin Pro Ala Ser Ala Lys Trp Tyr Asp Arg Arg Asp Tyr Val Phe 42 J281473 15 10 15 lie Glu Phe Cys Val Glu Asp Ser Lys Asp Val Asn Val Asn Phe Glu 20 25 30

Lys Ser Lys Leu Thr Phe Ser Cys Leu Gly Gly Ser Asp Asn Phe Lys 35 40 45

His Leu Asn Glu Ile Asp Leu Phe His Cys He Asp Pro Asn Asp Ser 50 55 60

Lys His Lys Arg Thr Asp Arg Ser lie Leu Cys Cys Leu Arg Lys Gly 65 70 75 80

Glu Ser Gly Gin Ser Trp Pro Arg Leu Thr Lys Glu Arg Ala Lys Leu 85 90 95

Asn Trp Leu Ser Val Asp Phe Asn Asn Trp Lys Asp Trp Glu Asp Asp 100 105 110

Ser Asp Glu Asp Met Ser Asn Phe Asp Arg Phe Ser Glu 115 120 125 43

Claims (1)

1281473 弘夸3月3日修(更)正本 拾、申請專利範圍： 1. 一種將生物標記分子用於檢測肝臟疾病之用途，其中前 述生物標記分子係具有SEQ ID N0:2、5、6、7、11、 12、13、14、15、18、20、23或24所示之任一胺基酸 序列，或前述胺基酸序列對應之自體抗體。 2. 如申請專利範圍第1項所述之用途，其中前述肝臟疾病 係為肝硬化或肝癌。 3. —種肝臟疾病之檢測套組，包含：生物標記分子，其係 選自 SEQ ID NO:2、5、6、7、11、12、13、14、15、 18、20、23及24所示之任一胺基酸序列。 4.如申請專利範圍第3項所述之肝臟疾病之檢測套組，其 中前述肝臟疾病係為肝硬化或肝癌。 5 ·如申請專利範圍第3項所述之肝臟疾病之檢測套組，其 中前述檢測套組係可進一步包含可辨識SEQ ID NO:2、 5、6、7、11、12、13、14、15、18、20、23 及 24 所示 之任一胺基酸序列之自體抗體之二級抗體。 6. —種肝臟疾病之篩檢方法，係包含下列步驟： 提供一檢體； 利用 SEQ ID NO:2、5、6、7、11、12、13、 14、15、18、20、23及24所示之任一胺基酸序列之生 物標記分子辨識捕捉檢體中之自體抗體；及 偵測自體抗體。 7. 如申請專利範圍第6項所述之篩檢方法，其中前述檢體 係包含全血或血清。 8. 如申請專利範圍第7項所述之篩檢方法，其中前述檢體 係為血清。 21 1281473 9.如申請專利範圍第6項所述之篩檢方法，其中前述生物 標記分子係可製成檢測套組。 10·如申請專利範圍第6項所述之篩檢方法，其中前述生物 標記分子係可進一步先行固定於基材上。 11.如申請專利範圍第10項所述之篩檢方法，其中前述基材 係為免疫分析盤或生物晶片。 12·如申請專利範圍第6項所述之篩檢方法，其中前述檢體 係可進一步先行以螢光標記進行標示。 13·如申請專利範圍第6項所述之篩檢方法，其中前述篩檢 方法係可增加一利用二級抗體辨識吸附自體抗體之步 驟。 14.如申請專利範圍第13項所述之篩檢方法，其中前述二級 抗體係經修飾具有特殊官能基可進行顯色反應、放射性 偵測或螢光偵測。 15·如申請專利範圍第6項所述之篩檢方法，其中前述偵測 自體抗體係利用螢光掃瞄分析偵測具有螢光標記之自體 抗體。 16. 如申請專利範圍第6項所述之篩檢方法，其中前述偵測 自體抗體係利用酵素連結免疫吸附分析（Enzyme-linked Immunosorbent Assay 5 ELISA )、放射免疫分析 (Radioimmunoassay，RIA )或免疫螢光分析 (Immunofluorescence )债測二級抗體。 17. —種肝臟疾病之檢測套組，包含一組可辨識具有SEQ ID NO:2、5、6、7、11、12、13、14、15、18、20、23 及 24所示之任一胺基酸序列之抗體。 18. 如申請專利範圍第17項所述之肝臟疾病之檢測套組，其 22 1281473 中前述肝臟疾病係為肝硬化或肝癌。 19. 一種肝臟疾病之篩檢方法，係包含下列步驟： 提供一檢體； 利用 SEQ ID N0:2、5、6、7、11、12、13、 14、15、18、20、23及24所示之任一胺基酸序列對應 之抗體辨識捕捉檢體中之抗原；及 偵測抗體與抗原之複合物。 20. 如申請專利範圍第19項所述之篩檢方法，其中前述檢體 係為全血或血清。 21. 如申請專利範圍第20項所述之篩檢方法，其中前述檢體 係為血清。

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